CN1997402B

CN1997402B - Cytotoxic compounds and conjugates

Info

Publication number: CN1997402B
Application number: CN200580020164.XA
Authority: CN
Inventors: S·勃艾德; L·陈; S·冈沃; V·古尔拉维斯; K·霍根; 李志宏; B·苏菲
Original assignee: Medarex LLC
Current assignee: E.R. expensive precious & Sheng Si limited liability company is executed
Priority date: 2004-05-19
Filing date: 2005-05-19
Publication date: 2014-04-02
Anticipated expiration: 2025-05-19
Also published as: CN1997402A; UA85716C2; ZA200609483B

Abstract

The present disclosure provides drug-ligand conjugates that are potent cytotoxins, wherein the drug is linked to the ligand through either a peptidyl, hydrazine, or disulfide linker. The disclosure is also directed to compositions containing the drug-ligand conjugates, and to methods of treatment using them.

Description

Cytotoxic compounds and conjugate thereof

U.S. Provisional Patent Application series the 60/572nd is enjoyed in the application's request, the rights and interests of 667,60/661,174 and 60/669, No. 871, and they are introduced into herein as a reference.

Invention field

The invention provides and be connected with part with medicine and the linking group of cracking in vivo.This linking group is useful in forming prodrug of the present invention and cytotoxin conjugate and other diagnosis and treatment part.

Background of invention

A lot of therapeutic agents, particularly in cancer chemotherapy, especially effectively those often show acute toxicity in body, especially bone marrow and mucosa toxicity, and chronic cardiac and neurological's toxicity.So high toxicity has limited their application.Developing more and safer specific treatment agent, particularly antitumor agent need to have larger effectiveness to tumor cell, and reduces quantity and the seriousness of the side effect (toxicity, to destruction of non-tumor cell etc.) of these products.The another kind difficulty of some existing therapeutic agent is that their stability in blood plasma is not ideal.The functional group adding in order to stablize these compounds causes active remarkable reduction.Therefore, need to determine the method that maintains acceptable therapeutic activity level in stable compound.

For the research of the cytotoxic agent of high selectivity more, very enliven in decades, the toxicity of dose limitation (the namely worthless activity of cytotoxin normal tissue) is the one of the main reasons of cancer therapy failure always.For example, known CC-1065 and duocarmycin SA (duocarmycins) are extremely effective cytotoxins.

CC-106 5 first by Upjohn Company in 1981 from Streptomyceszelensis separation (people such as Hanka, J.Antibiot.31:1211 (1978); The people such as Martin, J.Antibiot.33:902 (1980); The people such as Martin, J.Antibiot.34:1119 (1981)), and find in vitro with laboratory animal in have effective antitumour and antimicrobial acivity people such as (, Cancer Res.42:999 (1982)) Li.CC-1065 is combined the (people such as Swenson in ditch with double-stranded b form dna, Cancer Res.42:2821 (1982)), the sequence preference with 5 '-d (A/GNTTA)-3 ' and 5 '-d (AAAAA)-3 ', and the N3 position by CPI left hand unit the alkanisation 3 '-adenine in its molecule people such as (, Science 226:843 (1984)) Hurley.Although have effectively and anti-tumor activity widely, CC-1065 can not be for people, because it causes the laboratory animal that postpones dead.

A lot of analog and the derivant of CC-1065 and duocarmycin SA are known in the art.The structure of a lot of compounds, the research of synthetic and character have been summarized.Such as referring to people such as Boger, Angew.Chem.Int.Ed.Engl.35:1438 (1996); The people such as Boger, Chem.Rev.97:787 (1997).

Kyowa Hakko Kogya Co., a large amount of CC-1065 derivants have been prepared by the Yi Ge group of Ltd..For example, referring to United States Patent (USP) the 5th, 101,038,5,641,780,5,187,186,5,070,092,5,703,080,5,070,092,5,641,780,5,101,038,5,084, No. 468, disclosed PCT application WO 96/10405 and disclosed European application 0537575 A1.

Upjohn Company (Pharmacia Upjohn) also prepares the derivant of CC-1065 always energetically.For example, referring to United States Patent (USP) the 5th, 739,350,4,978,757,5,332,837 and 4,912, No. 227.

Also have research to concentrate on and develop new therapeutic agent, they are forms of prodrug, namely by their structure being carried out to some chemistry or zymetology, modify the compound that is converted into medicine (active treatment compound) in can body.For the object that reduces toxicity, this transformation is preferably limited to site of action or target tissue rather than blood circulation or non-target tissue.But, even if prodrug is also debatable, because due to the existence of enzyme, be much that the low stability of take in blood and serum is feature, these enzymes are degraded or activate prodrug before desired area in prodrug arrives patient body.

Bristol Laboratories Meyer is executed the antitumor drug conjugate your treasured (Bristol-Myers Squibb) has been described special lysosomal enzyme cleavable.Referring to for example United States Patent (USP) the 6th, 214, No. 345.This patent provides ammonia benzyl oxygen carbonyl.

Seattle Genetics has published No. 2003/0096743rd, U.S. Patent application and U.S. Patent application No. 2003/0130189, has wherein described the PAB ether in drug delivery agent.At the linking group described in these applications, only limit to aminobenzyl ether composition.

Other research group has also been described linking group.Referring to such as the people such as de Groot, J.Med.Chem.42,5277 (1999); The people J.Org.Chem.43 such as de Groot, 3093 (2000); The people such as de Groot, J.Med.Chem.66,8815, (2001); WO02/083180; The people such as Carl, J.Med.Chem.Lett.24,479, (1981); The people such as Dubowchik, Bioorg & Med.Chem.Lett.8,3347 (1998).These linking groups comprise that the electronics cascade of aminobenzyl ether spacer groups, prolongation and cyclisation spacer groups system, cyclisation eliminate spacer groups, for example amino amino carbonyl of w-, and PAB oxygen carbonyl linking.

The stability of cytotoxic drugs, comprises body internal stability, remains the major issue that needs solution.In addition, the toxicity of a lot of compounds makes them there is no function, therefore need to reduce the compositions that drug toxicity for example forms the prodrug of cleavable.Therefore, although certain progress has been obtained in this area, also still need to develop improved therapeutic agent, be used for the treatment of mammal, people particularly, therapeutic agent is more specifically cytotoxin, effect specificity, the toxicity of minimizing and the blood stability of raising that their performances for the known compound of analog structure are higher.The present invention is devoted to these demands.

Summary of the invention

The present invention relates to medicine-ligand conjugates, wherein, medicine is connected by peptidyl, hydrazine or disulphide linking group with part.These conjugates are to be delivered to site of action interested with activity form selectivity, and then cracking discharges the effective cytotoxin of active medicine.New linking group arm of the present invention can be in vivo mode cracking from cytotoxic drug by for example enzymatic lysis or reductive cleavage out, from prodrug derivant, discharge active medicine part.In addition, present invention resides in the conjugate between linking group arm of the present invention and cytotoxin, and the conjugate between linking group arm, cytotoxin and targeting agent, wherein, targeting agent is for example antibody or peptide.

The invention still further relates to the group that can be used for stablizing therapeutic agent and label.Stable group is chosen such that the enzyme that for example may be present in blood or non-target tissue with limit treatment agent or label removes and metabolism.Stable group can play the effect that stops medicine or label degraded, also can be used for providing other physical features of medicine or label, for example, increase the gathering character of dissolubility or the reduction compound of compound.Stable group also can improve medicine or label in the stability of lay up period, no matter is through the form of preparation or the form of not passing through preparation.

In first aspect, the invention provides the cytotoxic drug-ligand compound with arbitrary structure in any formula 1-3:

X ⁴

L ⁴ _pF L ¹

_m

D (1)

x ⁴

L ⁴ _pH

L ¹ _m D (2)

x ⁴

L ⁴ _pJ

L ¹

_m

D (3)

Wherein, symbol D has the drug moiety of pendent group in its main chain chemical reactivity functional group, and described functional group is selected from primary amine or secondary amine, hydroxyl, sulfydryl, carboxyl, aldehyde and ketone.

Symbol L ¹represent self sacrifice type (self-immolative) spacer groups, wherein, m is 0,1,2,3,4,5 or 6 integer.

Symbol X ⁴representative is selected from shielded reactive functional groups, not protected reactive functional groups, detectable labelling and targeting agent.

Symbol L ⁴represent linking group part, and p is 0 or 1.L ⁴conjugate dissolubility is improved or assemble the part that character reduces.L ⁴the example of part comprises assorted alkyl or the unsubstituted assorted alkyl of the aryl of the alkyl of replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, any one in them can be straight chain, side chain or ring-type, the amino acid polymer of lotus positive electricity or bear electricity, for example polylysine or poly arginine, or other polymer, for example Polyethylene Glycol.

Symbol F, H and J represent linking group as further described herein.

In one embodiment, the present invention relates to the peptide linking group conjugate of lower array structure:

X ⁴ (L ⁴) _p-F-(L ¹) _m D

Wherein,

D has the drug moiety of pendent group in its main chain chemical reactivity functional group, and described functional group is selected from primary amine or secondary amine, hydroxyl, mercaptan, carboxyl, aldehyde and ketone;

L ¹it is self sacrifice type linking group;

M is 0,1,2,3,4,5 or 6 integer;

F is the linking group that comprises lower array structure:

Or

Wherein,

AA ¹one or more groups independently selected from natural amino acid and non-natural a-amino acid;

C is 1 to 20 integer;

L ²it is self sacrifice type linking group;

L ³it is the spacer groups that comprises primary amine or secondary amine or carboxyl functional group; Wherein, if L ³exist, m is 0, and or L ³amine and the pendant carboxy group functional group of D form amido link, or L ³carboxyl and the suspension amine functional group of D form amido link;

O is 0 or 1;

L ⁴linking group part, wherein, L ⁴do not comprise direct and (AA ¹) _cthe connected carboxylic acyl radical of N-terminal;

P is 0 or 1; With

X ⁴be selected from the group of shielded reactive functional groups, not protected reactive functional groups, detectable labelling and targeting agent.

In one embodiment, peptide linking group conjugate comprises following structure:

In another embodiment, peptide linking group conjugate comprises following structure:

In preferred embodiments, L ³comprise aromatic group.For example, L ³can comprise benzoic acid group, aniline group or indolyl radical.-L ³the limiting examples of-NH-comprises and is selected from following structure:

Wherein, Z is selected from O, S and NR ²³, and

Wherein, R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.

In the preferred embodiment of peptide linking group, (AA ¹) _cit is the peptide sequence of the protease cracking that can be expressed in tumor tissues.Preferred protease is lysosomal protein enzyme.In preferred embodiments, c is 2 to 6 integer, or c is 2,3 or 4.In certain embodiments, (the AA nearest with drug moiety position ¹) _cin aminoacid be selected from: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.In preferred embodiments, (AA ¹) _cto be selected from following peptide sequence: Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, phe-N ⁹-tosyl-Arg, phe-N ⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO:1), β-Ala-Leu-Ala-Leu (SEQ ID NO:2) and Gly-Phe-Leu-Gly (SEQ ID NO:3).In particularly preferred embodiments, (AA ¹) _cval-Cit or Val-Lys.

In some preferred embodiments, peptide linking group, F, comprises following structure:

Wherein,

R ²⁴be selected from assorted alkyl and the unsubstituted assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement;

Each K is independently selected from the alkyl replacing, unsubstituted alkyl, the assorted alkyl of replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ²¹r ²², NR ²¹cOR ²², OCONR ²¹r ²², OCOR ²¹and OR ²¹

Wherein,

R ²¹and R ²²independently selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl; With

A is 0,1,2,3 or 4 integer.

In other preferred embodiment ,-F-(L ¹) _m-comprise following structure:

Wherein, each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement.

In one aspect of the method, the present invention relates to the hydrazine linking group conjugate of following structure:

X ⁴-(L ⁴) _p-H-(L ¹) _m-D

Wherein,

L ¹it is self sacrifice type linking group;

M is selected from 0,1,2,3,4,5 or 6 integer;

X ⁴be selected from shielded reactive functional groups, not protected reactive functional groups, detectable labelling and targeting agent;

L ⁴it is linking group part;

P is 0 or 1;

H is the linking group that comprises following structure:

Wherein, n ₁it is the integer from 1-10;

N ₂0,1 or 2;

Each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement; With

I or key, or:

Wherein, n ₃be 0 or 1, condition is to work as n ₃0 o'clock, n ₂not 0; And n ₄1,2 or 3,

Wherein, when I is key, n ₁3, and n ₂be 1 o'clock, D can not be

Wherein, R is Me or CH ₂-CH ₂-NMe ₂.

In some preferred embodiments, the replacement on benzyl ring is para-orientation.In some preferred embodiments, n ₁2,3 or 4, or n ₁3 or n ₂1.

In certain embodiments, I is key.In other embodiments, n ₃0 and n ₄2.

In aspect different, the invention provides hydrazine linking group, H, it can form 6-unit self sacrifice type linking group by cracking, or form two 5-unit self sacrifice type linking groups by cracking, or form the first self sacrifice type of single 5-linking group by cracking, or form the first self sacrifice type of single 7-linking group by cracking, or form 5-unit's self sacrifice type linking group and 6-unit self sacrifice type linking group by cracking.

In preferred embodiments, H comprises two dimethyl replacements.

In preferred embodiments, H comprises following structure:

Preferably, n ₁2,3 or 4, more preferably, n ₁3.Preferably, each R ₂₄independently selected from CH ₃and H.In some preferred embodiment, each R ₂₄h.

In a further preferred embodiment, H comprises following structure:

Preferably, n ₁3.Preferably, each R ²⁴independently selected from CH ₃and H.

In another preferred embodiment, H comprises following structure:

Preferably, each R ²⁴h or replacement or unsubstituted alkyl independently.

X ⁴-(L ⁴) _p-H-(L ¹) _m-D

Wherein,

L ¹it is self sacrifice type linking group;

M is selected from 0,1,2,3,4,5 or 6 integer;

L ⁴it is linking group part;

P is 0 or 1; With

H comprises following structure:

Wherein, q is 0,1,2,3,4,5 or 6; With

Each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement.

In a further aspect, the present invention relates to the disulphide linking group conjugate of lower array structure:

X ⁴

(L ⁴) _p-J-(L ¹) _m

D

Wherein,

L ¹it is self sacrifice type linking group;

M is selected from 0,1,2,3,4,5 or 6 integer;

L ⁴it is linking group part;

P is 0 or 1;

J is the linking group that comprises lower array structure:

Wherein,

Each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement;

Each K is independently selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ²¹r ²², NR ²¹cOR ²², OCONR ²¹r ²², OCOR ²¹and OR ²¹

Wherein,

R ²¹and R ²²heterocyclylalkyl and unsubstituted Heterocyclylalkyl independently selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, replacement;

A is 0,1,2,3 or 4 integer; With

D is 0,1,2,3,4,5 or 6 integer.

In different embodiments, J can comprise one of following structure:

Wherein, d is 1 or 2;

Or

In all above-mentioned linking group conjugates, D is cytotoxic drug preferably.In preferred embodiments, D has and is selected from following chemical reactivity functional group: primary amine or secondary amine, hydroxyl, sulfydryl and carboxyl.The limiting examples of preferred agents D comprises duocarmycin SA and duocarmycin SA analog and derivant, CC-1065, take CBI as basic duocarmycin SA analog, take MCBI as basic duocarmycin SA analog, take CCBI as basic duocarmycin SA analog, doxorubicin, doxorubicin conjugate, morpholino-doxorubicin, cyano group morpholino-doxorubicin, dolastatin, dolastatin-10, combretastatin, calicheamycin (calicheamicin), maitansine (maytansine), maitansine analog, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoyl valeric acid-AE ester (AEVB), tubulysins, disorazole, epothilones (epothilones), paclitaxel, Docetaxel, SN-38, hycamtin, rhizomycin, Quinomycin A., colchicine, vinblastine, vindesine, estramustine, Cemadotin, Eleutherobin. (eleutherobin), methotrexate, methopterin (methopterin), dichioromethotrexate, 5-fluorouracil, 6-MP, cytosine arabinoside, melphalan, leurosine (leurosine), inrosidine (leurosideine), D actinomycin D, daunorubicin, daunorubicin conjugate, ametycin, Mitomycin A, carubicin, aminopterin, talisomycin, podophyllotoxin, podophyllotoxin derivative, etoposide, etoposide phosphate, vincristine, taxol, taxotere tretinoin, butanoic acid, N ⁸-acetyl spermidine and camptothecine.

In preferred embodiments, D is duocarmycin SA analog or the derivant with lower array structure:

Wherein, ring system A is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl.

E and G are independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, hetero atom, singly-bound, or E and G be connected to form ring system, this ring system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl;

X is selected from O, S and NR ²³;

R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group;

R ³be selected from (=O), SR ¹¹, NHR ¹¹and OR ¹¹,

Wherein,

R ¹¹be selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, bisphosphate, triguaiacyl phosphate, acyl group, C (O) R ¹²r ¹³, C (O) OR ¹², C (O) NR ¹²r ¹³, P (O) (OR ¹²) ₂, C (O) CHR ¹²r ¹³, SR ¹²and SiR ¹²r ¹³r ¹⁴,

Wherein,

R ¹², R ¹³and R ¹⁴independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein, R ¹²and R ¹³together with the nitrogen-atoms connecting with them or carbon atom, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally contains two or more hetero atoms;

R ⁴, R ⁴', R ⁵and R ⁵' independently selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, SR ¹⁵, OR ¹⁵, CR ¹⁵=NR ¹⁶and O (CH ₂) _nn (CH ₃) ₂

Wherein

N is 1 to 20 integer;

R ¹⁵and R ¹⁶independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein, R ¹⁵and R ¹⁶together with the nitrogen-atoms connecting with them, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms;

R ⁶be singly-bound, it can exist also and can not exist, and works as R ⁶while existing, R ⁶and R ⁷be connected to form cyclopropyl rings; With

R ⁷cH ₂-X ¹, or in described cyclopropyl rings with R ⁶be connected-CH ₂-, wherein,

X ¹leaving group,

Wherein, R ¹¹, R ¹², R ¹³, R ¹⁵or R ¹⁶in at least one connect described medicine to L ¹, if present, or be connected to F, H or J.

In preferred embodiments, D has lower array structure:

Wherein,

Z is selected from O, S and NR ²³

Wherein,

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸, or CO ₂r ⁸, wherein, R ⁸be selected from the alkyl of replacement, unsubstituted alkyl, NR ⁹r ¹⁰, NR ⁹nHR ¹⁰and OR ⁹

Wherein

R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl; With

R ²alkyl or the unsubstituted low alkyl group of H, replacement;

In above preferred embodiment, R ²it is unsubstituted low alkyl group.

In a further preferred embodiment, D has following structure:

Wherein,

Z is selected from O, S and NR ²³

Wherein,

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸or CO ₂r ⁸, wherein, R ⁸be selected from NR ⁹r ¹⁰and OR ⁹,

Wherein,

R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl;

R ¹' be H, replacement or unsubstituted low alkyl group or C (O) R ⁸, wherein, R ⁸be selected from NR ⁹r ¹⁰and OR ⁹,

Wherein,

R ²h or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl or cyano group or alkoxyl; With

R ²' be H or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl,

In all above-mentioned linking group conjugate structures, L ⁴preferably comprise non-annularity part.With not containing L ⁴compound Phase ratio, L ⁴preferably increased the dissolubility of compound, and/or with not containing L ⁴compound Phase ratio, L ⁴reduced the gathering of compound.In preferred embodiments, L ⁴comprise polyalkylene glycol moiety.Polyalkylene glycol moiety for example can comprise, 3-12 recurring unit, and Huo2-6Ge recurring unit, or more preferably, 4 recurring units.

In a further aspect, the invention provides the cytotoxic drug-ligand compound with following formula structure:

X ⁴ L ⁴

_pQ L ¹ _mD ¹

Wherein, symbol L ¹represent self sacrifice type spacer groups, wherein, m is 0,1,2,3,4,5 or 6 integer.

Symbol X ⁴representative is selected from the member of shielded reactive functional groups, not protected reactive functional groups, detectable labelling and targeting agent.

Symbol L ⁴represent linking group part, and p is 0 or 1.L ⁴conjugate dissolubility is increased or assemble the part that character reduces.L ⁴the example of part comprises assorted alkyl or the unsubstituted assorted alkyl of the aryl of the alkyl of replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, any one in them can be straight chain, side chain or ring-type, the amino acid polymer of lotus positive electricity or bear electricity, for example polylysine or poly arginine, or other polymer is as Polyethylene Glycol.

Symbol Q represents the linking group of any cleavable, includes but not limited to any peptidyl as herein described, hydrazone and disulphide linking group.The linking group of cleavable comprises can be optionally from X ⁴by chemistry or biological process cracking with by cracking separate drug, D ¹, those.

Symbol D ¹representative has the medicine of following formula:

Wherein, X and Z are independently selected from O, S and NR ²³;

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸or CO ₂r ⁸,

R ¹' be H, replacement or unsubstituted low alkyl group or C (O) R ⁸,

Wherein, R ⁸be selected from NR ⁹r ¹⁰and OR ⁹, and R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl;

R ²h or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl or cyano group or alkoxyl;

R ³be selected from SR ¹¹, NHR ¹¹and OR ¹¹, wherein, R ¹¹be selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, bisphosphate, triguaiacyl phosphate, acyl group, C (O) R ¹²r ¹³, C (O) OR ¹², C (O) NR ¹²r ¹³, P (O) (OR ¹²) ₂, C (O) CHR ¹²r ¹³, SR ¹²and SiR ¹²r ¹³r ¹⁴, wherein, R ¹², R ¹³and R ¹⁴independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein, R ¹²and R ¹³the nitrogen or the carbon atom that together with them, connect, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, and this ring system optionally comprises two or more hetero atoms;

Wherein, R ¹¹, R ¹²and R ¹³in at least one connect described medicine to L ¹, if present, or be connected to Q,

R ⁶be singly-bound, it exists or does not exist, and when existing, R ⁶and R ⁷be connected to form cyclopropyl rings; With

R ⁷cH ₂-X ¹, or in described cyclopropyl rings, with R ⁶be connected-CH ₂-, wherein,

X ¹leaving group,

R ⁴, R ⁴', R ⁵and R ⁵' independently selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, SR ¹⁵, OR ¹⁵, CR ¹⁵=NR ¹⁶and O (CH ₂) _nnR ²⁴r ²⁵, wherein, n is 1 to 20 integer;

And R ²⁴and R ²⁵independently selected from unsubstituted alkyl, and

Wherein, at least one R ⁴, R ⁴', R ⁵and R ⁵' be O (CH ₂) _nnR ²⁴r ²⁵.

Another embodiment is the compound with formula 1 structure:

Wherein, X and Z are independently selected from O, S and NR ²³, wherein, R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group;

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸or CO ₂r ⁸,

R ¹' be H, replacement or unsubstituted low alkyl group or C (O) R ⁸,

Each R ⁸independently selected from NR ⁹r ¹⁰and OR ⁹, and R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl;

R ²h, replacement or unsubstituted low alkyl group, unsubstituted assorted alkyl, cyano group or alkoxyl;

R ²' be H, replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl,

R ³be selected from SR ¹¹, NHR ¹¹and OR ¹¹, wherein, R ¹¹be selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, bisphosphate, triguaiacyl phosphate, acyl group, C (O) R ¹²r ¹³, C (O) OR ¹², C (O) NR ¹²r ¹³, P (O) (OR ¹²) ₂, C (O) CHR ¹²r ¹³, SR ¹²and SiR ¹²r ¹³r ¹⁴, wherein, R ¹², R ¹³and R ¹⁴independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, or R ¹²and R ¹³together with the nitrogen connecting with them or carbon atom, form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms;

R ⁷cH ₂-X ¹, or in described cyclopropyl rings, with R ⁶be connected-CH ₂-, wherein, X ¹leaving group,

And R ²⁴and R ²⁵independently selected from unsubstituted alkyl, and

In a further aspect, the present invention relates to pharmaceutical preparation.Described preparation comprises compound and the pharmaceutically acceptable carrier puted together of the present invention conventionally.

In a still further aspect, the present invention relates to use method of puting together compound of the present invention.For example, the invention provides the method for cell killing, wherein, to cell, use the compound of puting together of the present invention of the amount that is enough to kill this cell.In preferred embodiments, cell is tumor cell.In another embodiment, the invention provides the method that delays or stop mammalian subject tumor growth, wherein, the compound of puting together of the present invention delays or stops the amount of tumor growth to patient's administration being enough to.

Other side of the present invention, advantage and object are by apparent the summary of the detail specifications from below.

The detailed description of invention

abbreviation

When for herein time, " Ala " refers to alanine.

" Boc " refers to tertbutyloxycarbonyl.

" CPI " refers to cyclopropane pyrrolo-indole.

" Cbz " is benzyloxycarbonyl group.

When for herein time, " DCM " refers to dichloromethane.

" DDQ " refers to 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone.

DIPEA is diisopropylethylamine

" DMDA " is N, N '-dimethyl-ethylenediamine

" RBF " is round-bottomed flask

" DMF " is N, B-dimethyl formamide

" HATU " is N-[[(dimethylamino)-1H-1,2,3-triazol [4,5-b] pyridine-1-yl] methylene]-N-methyl methanaminium hexafluorophosphate N-oxide

When for herein time, symbol " E " represents enzyme cleavable group.

" EDCI " is 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide.

When for herein time, " FMOC " refers to 9-fluorenylmethyloxycarbonyl.

" FMOC " refers to 9-fluorenylmethyloxycarbonyl.

" HOAt " is 7-azepine-I-hydroxybenzotriazole.

" Leu " is leucine.

" PABA " refers to para-amino benzoic acid.

PEG refers to Polyethylene Glycol

" PMB " refers to methoxy-benzyl.

" TBAF " refers to tetrabutylammonium fluoride.

Abbreviation " TBSO " refers to t-butyldimethylsilyl ether.

When for herein time, " TEA " refers to triethylamine.

" TFA " refers to trifluoroacetic acid.

Symbol " Q " refers to therapeutic agent, diagnostic agent or detectable labelling.

definition

Unless other definition, whole technology used herein and scientific terminology generally have the confessed implication of one skilled in the art of the present invention.Generally speaking, the laboratory operation in nomenclature used herein and following cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization is all well known in the art and conventional.Adopt standard technique to carry out the synthetic of nucleic acid and peptide.Generally speaking, enzyme reaction and purification step are to carry out according to the standard of manufacturer.Technology and operation be generally according to the conventional method in this area and various general list of references, carry out (generally referring to people such as Sambrook, Molecular Cloning:A Laboratory Manual, the 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., it is introduced into herein as a reference), these methods spread all over the document.Laboratory operation in nomenclature used herein and following analysis chemistry and organic synthesis is all well known in the art and conventional.Adopt standard technique or its improvement to carry out chemosynthesis and chemical analysis.

A kind of like this compound intended to represent in term " therapeutic agent ", when there is treatment effective dose, mammal produced to required therapeutic effect.For treatment cancer, need to can also enter target cell for this therapeutic agent.

Term " cytotoxin " plan represents a kind of like this therapeutic agent, and it has required cytotoxic effect to cancerous cell.Cytotoxicity refers to medicament to be stoped Growth of Cells or kills cell.--only supplying for example and not limitation--combretastatin class (combretastatins) that exemplary cytotoxin comprises, duocarmycin SA, CC-1065 antitumor antibiotics, anthracycline (anthracyclines) and related compound.Other cytotoxin comprises mycotoxin, ricin and analog thereof, calicheamycin, doxorubicin and CHROMATOGRAPHIC FRACTIONATION AND MASS (maytansinoids).

Term " prodrug " and term " drug conjugate " are used interchangeably in this article.The two all refers to cell relatively nontoxic and remain and put together form, but as being positioned at target cell or near enzyme target cell, by selectivity, is degraded into the compound of pharmacological activity form by some condition.

Term " label " intend to represent can be used for the compound of tumor or other medical condition characteristic differentiation, for example the progress of diagnosis, tumor and by the mensuration of the factor of tumor cell secretion.Label is regarded as the subset of " diagnostic agent ".

The heating rate that term " selectivity " refers to linking group part when combining use with enzymatic lysis product is greater than the heating rate containing the peptide of aminoacid random sequence.

Term " targeting group " and " targeting agent " intend to represent a kind of like this part, the entity (for example therapeutic agent or label) that its (1) can make it connect points to target cell, the tumor cell of specific type for example, or (2) preferentially target tissue for example tumor site be activated.Targeting group or targeting agent can be micromolecule, and its plan comprises non-peptide class and peptide class.Targeting group can also be macromole, comprises part, protein (such as BSA), antibody of sugar, phytohemagglutinin, receptor, receptor etc.In the preferred embodiment of the invention, targeting group is antibody or antibody fragment, more preferably monoclonal antibody or monoclonal antibody fragment.

Term " self sacrifice type spacer groups " refers to can be by the difunctional chemical part of two normally stable three part molecules of the covalently bound one-tenth of chemical part.Self sacrifice type spacer groups can spontaneous separation from second portion, if with the bond cleavage solution of first.

The part of detectable physics or chemical property intended to represent to have in term " detectable labelling ".

Unsettled part in vivo intended to represent in term " cleavable group ".Preferably, " cleavable group " allows label or therapeutic agent to be activated by cracking from conjugate remainder.Effectively definition is that linking group is preferably by cracking in biotic environment body.Cracking can be unrestrictedly from arbitrary process, such as enzyme, reproducibility, pH etc.Preferably, cleavable group is chosen such that so that activation occurs in required site of action, and this can be target cell (for example cancerous cell) or organization internal or near position, for example position of therapeutical effect or label activity.Such splitting action is the splitting action of enzyme, and exemplary enzyme cleavable group comprises natural amino acid or end at the peptide sequence of natural amino acid, at their carboxyl terminal, is connected with linking group.Although it is not key of the present invention that heating rate strengthens degree, but the preferred embodiment of cleavable linking group is such, wherein in administration 24 hours, in blood flow, cracking, at least about 10% cleavable group, most preferably is at least about 35%.

Term " part " refers to target cell or structural receptor, substrate, antigenic determinant or other binding site specific bond or associates reactively or compound any molecule.The example of part comprises antibody or its fragment (for example monoclonal antibody or its fragment), enzyme (for example plasmin), biological response modifier (for example interleukin, interferon, erythropeoitin or colony stimulating factor), peptide hormone and its Fab.

Term " hydrazine linking group " and " self-cyclisation hydrazine linking group " are used interchangeably in this article.These terms refer to by bar change part for example pH change and will carry out cyclization and form the linking group part of one or more rings.When contact, hydrazine Partial Conversion becomes hydrazone.For example can by with L ⁴there is this contact in the ketone group reaction in part.Owing to changing into hydrazone by contacting, therefore, term hydrazone linking group also can be for describing linking group of the present invention.

Term " five-first hydrazine linking group " or " 5-unit hydrazine linking group " refer to by condition Change Example and will carry out cyclization and form the molecular moiety containing hydrazine of one or more 5-rings as pH changes.Alternatively, these 5 yuan of linking groups can be described as five-first hydrazone linking group or 5-unit hydrazone linking group similarly.

Term " six-first hydrazine linking group " or " 6-unit hydrazine linking group " refer to by condition Change Example and will carry out cyclization and form the molecular moiety containing hydrazine of one or more 6-rings as pH changes.These 6 yuan of linking groups can be described as six-first hydrazone linking group or 6-unit hydrazone linking group similarly.

Term " cyclization ", when referring to the cyclisation of peptide, hydrazine or disulphide linking group, represents that linking group cyclisation ring formation, and starts to start the separation of medicine-ligand complex.This speed can be in the measurement of changing places, and when the product when at least 90%, 95% or 100% forms, it has just completed.

Term " polypeptide ", " peptide " and " protein " are used interchangeably in this article, represent the polymer of amino acid residue.These terms are applicable to such amino acid polymer, and wherein one or more amino acid residues are corresponding naturally occurring amino acid whose artificial chemistry analogies, are also applicable to the amino acid polymer that naturally occurring amino acid polymer and non-natural exist.Term " antibody also contained in these terms.”

Term " aminoacid " refers to naturally occurring and synthetic aminoacid, and amino acid analogue and amino acid analog thing, and the mode that they play a role is similar to naturally occurring aminoacid.Those that naturally occurring aminoacid is encoded by genetic code, and adorned those aminoacid, for example hydroxyproline, gamma carboxyglutamate and O-phosphoserine afterwards.Amino acid analogue represents such compound, they have the basic chemical structure identical with naturally occurring aminoacid, namely with α carbon, carboxyl, amino and the R group of hydrogen bonding, for example homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.Such analog has R group (for example nor-leucine) or the peptide backbone through modifying through modifying, but retains the basic chemical structure identical with naturally occurring aminoacid.Especially a spendable seed amino acid is citrulline, and it is arginine precursor, and participates in the formation of urea in liver.Amino acid analog thing refers to the compound with a kind of like this structure, and it is different from amino acid whose general chemical constitution, but the mode playing a role is similar to naturally occurring aminoacid.Term " alpha-non-natural amino acid " plan represents above-mentioned 20 kinds of naturally occurring amino acid whose " D " three-dimensional chemical configurations.Further be appreciated that term alpha-non-natural amino acid comprises the homologue of natural amino acid and the synthetic modification form of natural amino acid.Synthetic modification form includes but not limited to that alkylidene chain shortens or extends to aminoacid, the aminoacid that comprises optional substituted aryl of many two carbon atoms and comprise halo group, be preferably the aminoacid of haloalkyl and aryl.When being connected with linking group of the present invention or conjugate, aminoacid is the form of " amino acid side chain ", and wherein amino acid whose hydroxy-acid group is replaced by ketone group (C (O)).Thereby for example, alanine side chain is-C (O)-CH (NH ₂)-CH ₃, etc.

Aminoacid and peptide can be by blocking groups and protected.Blocking group is atom or the chemical part that protected amino acid or peptide N-end avoid carrying out undesirable reaction, and can in medicine-ligand conjugates building-up process, use.It will remain connected to N-end in whole building-up process, and can remove by the electrochemical conditions of removing or other condition that optionally realize it after being over drug conjugate is synthetic.The blocking group that is suitable for the protection of N-end is that chemistry of peptides field is known.Exemplary blocking group includes but not limited to hydrogen, D-aminoacid and benzyloxycarbonyl group (Cbz) chlorine.

" nucleic acid " represents the polymer of deoxyribonucleotide or ribonucleotide and strand or double chain form.This term is contained and is contained known nucleotide analog or the framework residue of process modification or the nucleic acid of key, they are that synthesize, naturally occurring and non-natural exists, there is the binding property similar to reference nucleic acid, by the mode of metabolism, be also similar to reference nucleotide.The example of this class analog unrestrictedly comprises thiophosphate, phosphoramidate, methyl phosphonate, chirality methyl phosphonate ester, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNAs).

Unless otherwise instructed, specific nucleotide sequence is for example also impliedly contained it, through conservative variant of modifying (codon of degenerating replaces) and complementary sequence and the sequence pointed out in detail.Particularly, the codon of degenerating replaces and can be realized by generating such sequence, the 3rd mixed alkali and/or the deoxyinosine residue of (or whole) codon that wherein one or more are selected replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991); The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); The people such as Rossolini, Mol.Cell Probes 8:91-98 (1994)).Term nucleic acid and gene, cDNA, mRNA, oligonucleotide and polynucleotide are used interchangeably.

Symbol

as key, still show that the upright position of a key all represents the point that the remainders such as shown part and molecule, solid carrier are connected.

Term " alkyl " represents--straight or branched or cyclic hydrocarbon atomic group or its combination unless specified otherwise herein--alone or as another substituent part, it can be completely saturated, single-or many-undersaturated, can comprise two-and many-valency atomic group, having specified carbon number (is C ₁-C ₁₀represent one to ten carbon).The example of saturated hydrocarbons atomic group includes but not limited to methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, such as the homologue of n-pentyl, n-hexyl, n-heptyl, n-octyl and isomer etc.Undersaturated alkyl is to have one or more pair of key or three key person.The example of undersaturated alkyl includes but not limited to vinyl, 2-acrylic, crotyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(Isosorbide-5-Nitrae-pentadienyl), acetenyl, 1-and 3-propinyl, 3-butynyl and higher homologue and isomer.Term " alkyl ", unless explained in addition, also means those alkyl derivatives that comprise following specific definition, for example " assorted alkyl ".The alkyl that is limited to hydro carbons group also claims " same alkyl ".

Term " alkylidene " represents from the derivative bivalent group of alkane alone or as another substituent part, such as but not limited to-CH ₂cH ₂cH ₂cH ₂-, further comprise those groups of following " assorted alkylidene ".Conventionally, alkyl (or alkylidene) will have 1 to 24 carbon atom, have 10 or still less those groups of carbon atom be preferred in the present invention." low alkyl group " or " low-grade alkylidene " is short-chain alkyl or alkylidene, generally has eight or carbon atom still less.

--stable straight or branched or cyclic hydrocarbon atomic group or its combination unless specified otherwise herein--that term " assorted alkyl " is combined expression alone or with another term, carbon atom and at least one hetero atom by specified quantity form, the group that hetero atom selects free O, N, Si and S to form, wherein nitrogen, carbon and sulphur atom are optionally oxidized, and nitrogen heteroatom is optionally quaternized.Hetero atom O, N, S and Si can be positioned at any interior location of assorted alkyl or the position that alkyl is connected with molecule remainder.Include but not limited to-CH of example ₂-CH ₂-O-CH ₃,-CH ₂-CH ₂-NH-CH ₃,-CH ₂-CH ₂-N (CH ₃)-CH ₃,-CH ₂-S-CH ₂-CH ₃,-CH ₂-CH ₂,-S (O)-CH ₃,-CH ₂-CH ₂-S (O) ₂-CH ₃,-CH=CH-O-CH ₃,-Si (CH ₃) ₃,-CH ₂-CH=N-OCH ₃with-CH=CH-N (CH ₃)-CH ₃.Two hetero atoms can be continuous at the most, for example-CH ₂-NH-OCH ₃with-CH ₂-O-Si (CH ₃) ₃.Similarly, term " assorted alkylidene " represents from the derivative bivalent group of assorted alkyl alone or as another substituent part, such as but not limited to-CH ₂-CH ₂-S-CH ₂-CH ₂-and-CH ₂-S-CH ₂-CH ₂-NH-CH ₂-.About assorted alkylidene, hetero atom also can occupy one or two chain end (such as alkylene oxide group, alkylene dioxo base, alkylene amino, alkylene diaminourea etc.).Term " assorted alkyl " and " assorted alkylidene " are contained PEG and derivant (for example, referring to Shearwater polymer Catalog, 2001) thereof.Further, for alkylidene and assorted alkylidene linking group, the direction that linking group structural formula is write does not also mean that the orientation of linking group.For example, formula-C (O) ₂r '-representative-C (O) ₂r '-and-R ' C (O) ₂-.

Represent to have the part of 1 to 6 carbon atom with term " alkyl " or " assorted alkyl " term " rudimentary " of combining.

Term " alkoxyl ", " alkylamino ", " alkyl sulphonyl " and " alkylthio group " (or thio alkoxy) are used their conventional sense, represent respectively via oxygen atom, amino SO ₂those alkyl that group or sulphur atom are connected with molecule remainder.Term " aryl sulfonyl " refers to via SO ₂the aryl that group is connected with molecule remainder, term " sulfydryl " refers to SH group.

Generally speaking, " acyl substituent " is also selected from above-mentioned group.Term used herein " acyl substituent " represents be connected with carbonyl carbon and meet its valent group, and this carbonyl carbon is directly or indirectly connected with the multi-ring core of the compounds of this invention.

Term " cycloalkyl " and " Heterocyclylalkyl " be alone or combine respectively representative with other term--to be replaced or the ring-type modification of unsubstituted " alkyl " and replacement or unsubstituted " alkyl of mixing " unless specified otherwise herein--.In addition, about Heterocyclylalkyl, hetero atom can occupy the position that heterocycle is connected with molecule remainder.The example of cycloalkyl includes but not limited to cyclopenta, cyclohexyl, 1-cyclohexenyl group, 3-cyclohexenyl group, suberyl etc.The example of Heterocyclylalkyl includes but not limited to 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, Tetramethylene sulfide-2-base, Tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl etc.The hetero atom of circulus and carbon atom are optionally oxidized.

Term " halo " or " halogen " represent alone or as another substituent part--fluorine, chlorine, bromine or iodine atom unless specified otherwise herein--.In addition, the term such as " haloalkyl " means and comprises single haloalkyl and multi-haloalkyl.For example, term " halo (C ₁-C ₄) alkyl " mean and include but not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl etc.

Term " aryl " represents, and--replace unless specified otherwise herein--or unsubstituted, polyunsaturated, aromatic hydrocarbon substituent group, it can be single ring or a plurality of condensing together or covalently bound ring (preferably 1 to 3 ring).Term " heteroaryl " represents to contain one to four heteroatomic aryl (or ring) that is selected from N, O and S, and wherein nitrogen, carbon and sulphur atom are optionally oxidized, and nitrogen-atoms is optionally quaternised.Heteroaryl can be connected with molecule remainder by hetero atom.The limiting examples of aryl and heteroaryl comprises phenyl, 1-naphthyl, 2-naphthyl, 4-xenyl, 1-pyrrole radicals, 2-pyrrole radicals, 3-pyrrole radicals, 3-pyrazolyl, 2-imidazole radicals, 4-imidazole radicals, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridine radicals, 3-pyridine radicals, 4-pyridine radicals, 2-pyrimidine radicals, 4-pyrimidine radicals, 5-benzothiazolyl, purine radicals, 2-benzimidazolyl, 5-indyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.The substituent group of each above-mentioned aryl and heteroaryl ring system is selected from following acceptable substituent group." aryl " and " heteroaryl " also contains such ring system, and wherein one or more non-aromatic ring systems and aryl or heteroaryl system condense or other bonding.

For simplicity, term " aryl " (for example aryloxy group, arylthio, aralkyl) when combining use with other term comprises aryl and heteroaryl ring as defined above.Thereby, term " aralkyl " means and comprises such atomic group, wherein aryl is connected (such as benzyl, phenethyl, pyridylmethyl etc.) with alkyl, this alkyl comprises such alkyl, and wherein carbon atom (such as methylene) is such as being replaced (such as phenoxymethyl, 2-pyridyloxy methyl, 3-(1-naphthoxy) propyl group etc.) by oxygen atom.

Above each term (for example " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") comprise shown in replacement and the unsubstituted form of atomic group.The preferred substituents of every type of atomic group is provided below.

The substituent group of alkyl and assorted alkyl atomic group (comprising those groups that are often called as alkylidene, alkenyl, assorted alkylidene, heterochain thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) is generally called " alkyl substituent " and " alkyl substituent of mixing, they can be selected from but be not limited to one or more of following various groups :-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R ,-OC (O) R ' ,-C (O) R ' ,-CO ₂r ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R

,-NR " C (O) ₂r ' ,-NR-C (NR ' R " R )=NR " " ,-NR-C (NR ' R ")=NR ,-S (O) R ' ,-S (O) ₂r ' ,-S (O) ₂nR ' R " ,-NRSO ₂r ' ,-CN and-NO ₂, quantitative range is from 0 to (2m '+1), and wherein m ' is the sum of carbon atom in this class atomic group.R ', R ", R

and R " " preferably represent independently separately hydrogen, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl--the aryl, replacement or unsubstituted alkyl, alkoxyl or thio alkoxy or the aralkyl that for example by 1-3 halogen, are replaced.When the compounds of this invention comprises more than one R group, for example each R group is selected independently, when there being more than one R ', R ", R

and R " " during group, they are also like this.As R ' and R " when identical nitrogen-atoms is connected, they can be combined with nitrogen-atoms and form 5-, 6-or 7-ring.For example ,-NR ' R " mean and include but not limited to 1-pyrrolidinyl and 4-morpholinyl.Those skilled in the art will appreciate that from above-mentioned substituent discussion, and term " alkyl " means the group that comprises the group bonding beyond wherein carbon atom and dehydrogenation, for example haloalkyl (for example-CF ₃with-CH ₂cF ₃) and acyl group (for example-C (O) CH ₃,-C (O) CF ₃,-C (O) CH ₂oCH ₃deng).

Be similar to about the substituent group described in alkyl atomic group, the substituent group of aryl and the substituent group of heteroaryl are generally called " aryl substituent " and " heteroaryl substituent group ", and be different, for example, be selected from: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R ,-OC (O) R ' ,-C (O) R ' ,-CO ₂r ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R

,-NR " C (O) ₂r ' ,-NR-C (NR ' R ")=NR ,-S (O) R ' ,-S (O) ₂r ' ,-S (O) ₂nR ' R " ,-NRSO ₂r ' ,-CN and-NO ₂,-R ' ,-N ₃,-CH (Ph) ₂, fluoro (C ₁-C ₄) alkoxyl and fluoro (C ₁-C ₄) alkyl, quantitative range is fastened and is opened valent sum from zero to aromatic ring; Wherein R ', R ", R

and R " " preferably independently selected from hydrogen, (C ₁-C ₈) alkyl and assorted alkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C ₁-C ₄) alkyl and (unsubstituted aryl) oxygen base-(C ₁-C ₄) alkyl.When the compounds of this invention comprises more than one R group, for example each R group is selected independently, when there being more than one R ', R ", R and R " " during group, they are also like this.

On the adjacent atom of aryl or heteroaryl ring two aryl substituents can be optionally by formula-T-C (O)-(CRR ') _q-U-substituent group replaces, and wherein T and U are-NR-,-O-,-CRR '-or singly-bound independently, and q is 0 to 3 integer.Alternatively, on the adjacent atom of aryl or heteroaryl ring, two substituent groups can be optionally by formula-A-(CH ₂) _r-B-substituent group replaces, wherein A and B be independently-CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O) ₂-,-S (O) ₂nR '-or singly-bound, r is 1 to 4 integer.One of singly-bound of the new ring so forming can optionally be replaced by two keys.Alternatively, on the adjacent atom of aryl or heteroaryl ring two substituent groups can be optionally by formula-(CRR ') _s-X-(CR " R ) _d-substituent group replaces, and wherein s and d are 0 to 3 integer independently, X is-O-,-NR '-,-S-,-S (O)-,-S (O) ₂-or-S (O) ₂nR '-.Substituent R, R ', R " and R preferably independently selected from hydrogen or replacement or unsubstituted (C ₁-C ₆) alkyl.

When for herein time, the ester of the phosphoric acid that term " bisphosphate " includes but not limited to comprise two phosphates.The ester of the phosphoric acid that term " triguaiacyl phosphate " includes but not limited to comprise three phosphates.For example, the concrete medicine containing bisphosphate or triguaiacyl phosphate comprises:

Term used herein " hetero atom " comprises oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

Symbol " R " is to represent substituent general abbreviation, and substituent group is selected from and replaces or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted heterocyclic radical.

Term " pharmaceutically acceptable carrier ", when for herein time, refers to the acceptable material of pharmacy, compositions or the carrier that participate in transportation or transhipment chemical agent, for example liquid filling agent or solid-filling agent, diluent, excipient, solvent or coating material.Pharmaceutically acceptable carrier comprises the acceptable salt of pharmacy, and wherein, term " the acceptable salt of pharmacy " comprises the salt of reactive compound, and they utilize relatively avirulent acid or alkali to prepare, and this depends on the specified substituent on compound described herein.When the compounds of this invention contains relatively acid functional group, can obtain like this base addition salts, the neutral form of this compounds is contacted, purely or in applicable atent solvent with enough required alkali.The example of the acceptable base addition salts of pharmacy comprises sodium, potassium, calcium, ammonium, organic amino or magnesium salt, or similar salt.When the compounds of this invention contains relatively the functional group of alkalescence, can obtain like this acid-addition salts, the neutral form of this compounds is contacted, purely or in applicable atent solvent with enough required acid.The example of the acceptable acid-addition salts of pharmacy comprises those that derive from mineral acid, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, a hydrogen carbonic acid, phosphoric acid, a hydrogen phosphoric acid, dihydrogen phosphoric acid, sulphuric acid, a hydrosulphuric acid, hydroiodic acid or phosphoric acid etc. of acid, and from the derivative salt of relatively avirulent organic acid, such as acetic acid, propanoic acid, isopropylformic acid., maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-methyl benzenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid etc. of acid.Also comprise amino acid whose salt, such as arginine salt etc., and the organic acid salt such as glucuronic acid or galacturonic acid is (such as referring to people such as Berge, " Pharmaceutical Salts ", Journal of Pharmaceutical Science, 1977,66,1-19).Some particular compound of the present invention contains alkalescence and acidic functionality, allows compound to be converted into alkali or acid-addition salts.

The neutral form of compound is regeneration so preferably, makes salt and alkali or sour contact, more separated parent compound in the usual way.The parent form of compound is different from various salt forms in some physical property, the dissolubility in polar solvent for example, but for purposes of the present invention, salt is equal to the parent form of compound.

Except salt form, the invention provides the compound of prodrug forms.The prodrug of compound described herein is such compound, and they easily experience chemical change under physiological condition, and compound of the present invention is provided.In addition, prodrug can be converted into compound of the present invention by chemistry or biochemical method in vitro environment.For example, when being placed in the transdermal patch Drug Storage that contains applicable enzyme or chemical reagent, prodrug can be converted into compound of the present invention lentamente.

Can there is the not form of solvation and the form of solvation in some the compounds of this invention, comprise the form of hydration.Generally speaking, the form of solvation is equal to the not form of solvation, all contains within the scope of the invention.Can there is polymorphic or amorphous in some the compounds of this invention.Generally speaking, all physical form are all equal in purposes involved in the present invention, all belong to scope of the present invention.

Some the compounds of this invention has asymmetric carbon atom (rotophore) or two key; Racemate, diastereomer, geometric isomer and individual other isomer are all contained within the scope of the invention.

The atom isotope that compound of the present invention can also contain non-natural part on the one or more atoms that form this compounds.For example, compound can be used radiosiotope radioactive label, for example tritium ( ³h), iodine-125 ( ¹²⁵i) or carbon-14 ( ¹⁴c).Whether all isotopic variations of the compounds of this invention are that radioactive all plans are contained within the scope of the invention.

Term " coupling part " or " part that connects targeting group " represent the part that allows targeting group to be connected with linking group.Typical linking group comprises, and--only unrestricted for setting forth--alkyl, aminoalkyl, amino carbonyl alkyl, carboxyalkyl, hydroxy alkyl, alkyl-maleimide, alkyl-N-hydroxy-succinamide, PEG-maleimide and PEG-N-hydroxy-succinamide, they all can be further substituted.Linking group can also make coupling part in fact invest targeting group.

Term used herein " leaving group " is illustrated in reaction the substrate part of cracking from substrate.

Term used herein " antibody " comprises whole antibody and any Fab (i.e. " antigen-bound fraction ") or its strand." antibody " refers to and comprises by disulphide bond or interconnected at least two weights (H) chain of its antigen-binding portion thereof and the glycoprotein of two light (L) chains.Every heavy chain comprises variable region of heavy chain (V _h) and CH.CH comprises three territories, C _h1, C _h2and C _h3, and can be μ, δ, γ, α or ζ isotype.Each light chain comprises variable region of light chain (V _l) and constant region of light chain.Constant region of light chain comprises Yi Ge district, C _l, it can be κ or λ isotype.V _hand V _ldistrict can further be subdivided into hypervariable region, and it is also referred to as complementary determining region (CDR), is wherein dispersed with the more conservative region that is called as framework region (FR).Each V _hand V _lby three CDR and four FR, formed, from amino terminal, be aligned to carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The variable region of heavy chain and light chain comprises the land with AI.The constant region of antibody can mediated immunity globulin and the combination of host tissue or the factor, comprises first composition (Clq) of immune different cell (for example effector lymphocyte) and classical complement system.

" antigen-bound fraction " of term " antibody fragment " or antibody (or simple " antibody moiety ") is when referring to the one or more antibody fragments that retain with antigenic specificity binding ability for herein time.Show, the antigen-combined function of antibody can complete by full length antibody fragment.The example of the binding fragment comprising at " antigen-bound fraction " of term " antibody fragment " or antibody comprises: (i) Fab fragment, and by V _l, V _h, C _land C _h1the unit price fragment that district forms; (ii) F (ab ') ₂fragment, the bivalence fragment that comprises two Fab fragments that are connected in hinge region by disulphide bridges; (iii) by V _hand C _h1the Fd fragment that district forms; (iv) by the V of antibody single armed _land V _hthe Fv fragment that district forms; (v) dAb fragment (people such as Ward, (1989) Nature 341: 544-546), it is by V _hdistrict forms; (vi) complementary determining region of separating (CDR).In addition, although Fv fragment Liang Ge district, V _land V _h, by independent gene code, but they can be connected by synthetic linking group by the method for restructuring, and this linking group can make them become single protein chain, wherein, V _land V _hdistrict is to forming monovalent molecule (also referred to as scFv (scFv); Referring to such as the people such as Bird (1988) Science 242: 423-426; With people (1988) Proc.Nat1.Acad.Sci.USA such as Huston 85: 5879-5883).Within such single-chain antibody also intends to be included in " antigen-bound fraction " of term antibody.Use routine techniques well known by persons skilled in the art to obtain these antibody fragments, screening fragment, usings and uses as complete antibody by same way.

Term " monoclonal antibody " refers to the goods of the antibody molecule of unimolecule compositions when using in this article.Monoclonal antibody combination has demonstrated single binding specificity and the affinity to specific antigen epi-position.

In order to prepare monoclonal or polyclonal antibody, can use the known any technology of prior art (referring to for example Kohler & Milstein, Nature 256:495-497 (1975); The people such as Kozbor, Immunology Today 4:72 (1983); The people MONOCLONALANTIBODIES AND CANCER THERAPY such as Cole, Alan R.Liss, the 77-96 page in Inc. (1985)).

The production method of polyclonal antibody is well known by persons skilled in the art.Inbred mice (for example BALB/C mice) or rabbit are caused to immunity with protein, use the adjuvant of standard, for example Freund ' s adjuvant, and standard cause immunization protocol.Animal is monitoring like this to the immunne response of immunogen prepared product, carries out tentative blood-letting, measures the reactivity of β subunit is tired.When obtaining suitably high antibody to immunogenic tiring, gather the blood of animal, prepare antiserum.Can carry out sero-fast further separation if necessary, with enrichment, protein is to reactive antibody.

The various technology that monoclonal antibody can be familiar with by those skilled in the art obtain.In brief, make extremely not changed by the splenocyte of the immunogenic animal of required antigen, generally by merging (Kohler & Milstein, Eur.J.Immunol.6:511-519 (1976)) with myeloma cell.The alternative method of extremely not changing comprises with Epstein-Barr virus, oncogene or retrovirus, or other method well known in the art.

In preferred embodiments, antibody is chimeric antibody or humanized antibody.Chimeric antibody of the present invention or humanized antibody can be take mouse monoclonal antibody sequence as basis preparation.The DNA of encoding heavy chain and light chain immunoglobulin can obtain from interested murine hybridoma, and is designed to comprise non-muroid (for example mankind) immunoglobulin sequences with standard molecular biological technique.For example, in order to manufacture chimeric antibody, use methods known in the art Mus variable region can be connected to human constant region (referring to No. the 4th, 816,567, the United States Patent (USP) such as authorizing the people such as Cabilly).In order to manufacture humanized antibody, use methods known in the art can Jiang Shu CDR district insert people's framework (referring to the United States Patent (USP) the 5th of for example authorizing Winter, 225, No. 539 and authorize the people's such as Queen United States Patent (USP) the 5th, 530,101,5,585,089,5,693,762 and 6,180, No. 370).

In a further preferred embodiment, antibody is people's antibody.Can produce this people's antibody by immune transgenic or transchromosomic mice, in this mice, endogenous mouse immunoglobulin genes is inactivated, and exogenous human immunoglobulin gene is introduced into.This mice is that prior art is known (referring to for example United States Patent (USP) the 5th, 545,806,5,569,825,5,625,126,5,633,425,5,789,650,5,877,397,5,661,016,5,814,318,5,874,299 and 5,770, No. 429, all these patents are all authorized Lonberg and Kay; Authorize the people's such as Kucherlapati United States Patent (USP) the 5th, 939,598,6,075,181,6,114,598,6,150,584 and 6,162, No. 963; PCT communique WO 02/43478 with people such as Ishida).People's antibody also can be with preparing for screening the phage display method in human immunoglobulin gene library.This phage display method for separating of people's antibody is also that prior art is known (referring to the United States Patent (USP) the 5th, 223,409,5,403,484 and 5,571 such as authorizing the people such as Ladner, No. 698; Authorize the people's such as Dower United States Patent (USP) the 5th, 427,908 and 5,580, No. 717; Authorize the people's such as McCafferty United States Patent (USP) the 5th, 969,108 and 6,172, No. 197; With the United States Patent (USP) the 5th, 885,793,6,521,404,6,544,731,6,555,313,6,582,915 and 6,593 of authorizing the people such as Griffiths, No. 081).

" solid carrier " used herein represents a kind of like this material, and it is insoluble to selected solvent system substantially, or can easily separation (for example passing through the sedimentation method) from selected suitable solvents system.Can be used for implementing solid carrier of the present invention and can comprise group activation or that can activate, to allow selected kind to be combined with solid carrier.Solid carrier can also be a kind of substrate, for example chip, wafer or hole, and individuality of the present invention or more than one compounds are in conjunction with on it.

" reactive functional groups " used herein represents such group, include but not limited to alkene, alkynes, alcohol, phenol, ether, oxide, halogenide, aldehyde, ketone, carboxylic acid, ester, amide, cyanate, isocyanates, rhodanate, isothiocyanate, amine, hydrazine, hydrazone, hydrazides, diazonium, diazol, nitro, nitrile, sulfur alcohol, sulfide, disulphide, sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal, ketal, anhydride, sulfate, sulfenic acids, isonitrile, amidine, sub-amide, imines hydrochlorate, nitrone, azanol, oxime, hydroxamic acid, sulfo-hydroxamic acid, allene, ortho esters, sulphite, enamine, ynamine, urea, pseudo-urea, semicarbazides, carbodiimide, carbamate, imines, azide, azo-compound, azoxy compound and nitroso compound.Reactive functional groups also comprises for the preparation of those of bioconjugates, (for example, referring to Hermanson, BIOCONJUGATETECHNIQUES, Academic press, San Diego, 1996) such as N-hydroxy-succinamide ester, maleimide.The method of preparing every kind of these functional groups is well known in the art, they are applied to specific object or revise all in those skilled in the art's limit of power (for example, referring to Sandler andKaro according to specific object, eds. organo-functional group preparation, Academic Press, San Diego, 1989).Reactive functional groups can be protected or not protected.

Compound of the present invention is made into the mixture of single isomer (for example enantiomer, cis-trans, position, diastereomer) or isomer.In preferred embodiments, compound is made into substantially single isomer.The method of preparing the compound of basic isomer-pure is known in the art.For example, the mixture of enrichment enantiomer and pure enantiomeric compounds can be prepared like this, the combination that utilizes the synthetic intermediate of enantiomer-pure to react with this class, and it is constant that these reactions maintain the spatial chemistry of chiral centre, or cause it to reverse completely.Alternatively, end-product or synthetic intermediate on the way can be split as single stereoisomer.For reversing specific Stereocenter or maintain its constant technology and be well known in the art for splitting the technology of stereoisomer mixture, those skilled in the art select suitable method according to specific situation completely in its limit of power.Generally referring to the people such as Furniss (editor), VOGEL ' SENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY the 5th edition, LongmanScientific and Technical Ltd., Essex, 1991, the 809-816 pages; Heller, Acc.Chem.Res.23:128 (1990).

linking group

The invention provides medicine-ligand conjugates, wherein, medicine is connected with part by cytotoxic compounds.This linking group is peptidyl, hydrazine or disulphide linking group, and they are described as respectively (L in this article ⁴) _p-F-(L ¹) _m, (L ⁴) _p-H-(L ⁱ) _mor (L ⁴) _p-J-(L ⁱ) _m.Except the linking group being connected with medicine, the present invention also provides the linking group arm being applicable to the cleavable that any molecule type is connected substantially.Linking group arm of the present invention makes an explanation with reference to it and being connected for the treatment of part in this article.Yet, it will be obvious to those skilled in the art that linking group can be connected to different kinds, include but not limited to diagnostic agent, analytical reagent, biomolecule, targeting agent and detectable labelling etc.

One aspect of the present invention relates to the linking group that can be used for connecting targeting group and therapeutic agent and label.The present invention provides such linking group on the other hand, and they give compound with stability, reduces their toxicity in vivo, or advantageously affects their pharmacokinetics, bioavailability and/or pharmacodynamics.Generally preferably in this class embodiment, once medicine is released into its site of action, linking group is cleaved, discharges active medicine.Thereby In one embodiment of the present invention, linking group of the present invention is traceless, once for example, to remove (between pot-life) from therapeutic agent or label, no longer there is the vestige of linking group.

In another embodiment of the invention, linking group be take them in target cell or near the cleaved ability in position be feature, the position of therapeutical effect or label activity for example.This class splitting action is actually the effect of enzyme.This specific character contributes to reduce the whole body activation of therapeutic agent or label, reduces toxicity and systemic side effects.The preferred cleavable group of enzymatic lysis product comprises peptide bond, ester bond and disulphide bond.In other embodiments, linking group is responsive to pH, and changes and cracking by pH.

Importance of the present invention be can the cracking of control connection group speed.For example, hydrazine linking group as herein described is useful especially, because according to ad hoc structure used, people can change the ring formation speed of linking group, thereby from part, cracking is out by medicine.WO02/096910 provides several part-medicinal compositions containing hydrazine linking group.Yet, according to required ring formation speed " coordination " linking group of having no idea, form, and described specific compound with respect to a lot of medicine-linking group conjugates preferably slower speed from medicine cracking part.On the contrary, hydrazine linking group of the present invention provides the ring formation speed of certain limit, from very near very slow, thereby according to required ring formation speed, can select specific hydrazine linking group.For example, can use the very fast ring formation of hydrazine linking group realization that produces single 5-ring by cracking.Use hydrazine linking group to realize the being preferably speed to cell by cytotoxic agent targeted delivery, because linking group has two methyl in paired position, this hydrazine linking group has produced two 5-rings or single 6-ring by cracking.With in paired position, do not have the single 6-ring of two methyl to compare, two dimethyl effects have demonstrated has accelerated cyclization speed.This is the tension alleviating due in ring.Yet sometimes, the substituent group reaction of can slowing down, rather than make its quickening.The reason of retardance usually can be followed the trail of to steric hindrance.As shown in embodiment 2.4, with respect to two carbon, be CH ₂, two dimethyl replace and have caused faster cyclization generation.

Yet, important being noted that in some embodiments, the preferably slower linking group of cracking.For example, in sustained release forms, or in the dosage form that not only contains rapid release but also form containing slow release, it may be useful that the slower linking group of cracking is provided.In certain embodiments, use hydrazine linking group to realize slower ring formation speed, this hydrazine linking group has produced single 6-ring by cracking, in the situation that not having two dimethyl to replace, or has produced single 7-ring.

Linking group also plays the effect of stablizing therapeutic agent or label, the Degradation of opposing in circulation.This specific character provides significant benefit, because the medicine that this class Stabilization prolongation connects or the circulating half-life of label.Linking group also plays the effect that weakens connected medicine or label activity, so that conjugate is relatively optimum in circulation, and has required effect after required site of action activation, for example, be toxicity.About therapeutic agent conjugate, this specific character of linking group plays the effect that improves Drug therapy index.

Stable group is preferably chosen such that the enzyme that may be present in blood or non-target tissue with limit treatment agent or label cleans up and metabolism, and is further chosen such that with limit drug or label transhipment and enters cell.Stable group plays the effect of blocking medicine or label degraded, and other physical features of medicine or label can also be provided.Stable group can also improve medicine or label in the stability of lay up period, is no matter through the form of preparation or through the form of preparation.

Ideally, if stablize the effect that group plays protection medicine or label degraded, so when medicine or label are stored after 2 hours in 37 ℃ of human bloods, cause being less than 20%, be preferably less than 10%, be preferably less than 5%, the medicine more preferably less than 2% or label be present in the enzymatic lysis in human blood under given condition determination, illustrates that it can be used for stablizing therapeutic agent or label.

The invention still further relates to the conjugate that contains these linking groups.More properly, the present invention relates to be used for the treatment of disease, the especially prodrug of cancer chemotherapy method.Particularly, the use of linking group described herein provides the blood stability with respect to the effect specificity of the prodrug Yan Genggao of analog structure, the toxicity of minimizing and raising for prodrug.

Linking group of the present invention as herein described can be present in any position in peptide-cytotoxic conjugates.

Thereby the linking group providing can contain any various group as a part for its chain, they are by cracking in vivo, and for example, in blood flow, speed is higher than the construct that lacks this class group.The conjugate of linking group arm and therapeutic agent and diagnostic agent is also provided.Linking group can be used for generating the prodrug analog of therapeutic agent, reversibly connects treatment or diagnostic agent and targeting agent, detectable labelling or solid carrier.Linking group can be combined into and comprise the cytotoxic coordination compound of the present invention.

Except peptide, hydrazine or the disulphide group of cleavable, the one or more self sacrifice type linking group L of optional introducing between cytotoxin and targeting agent ¹.These linking groups also can be described as spacer groups, and comprise at least two reactive functional groups.Typically, chemical functional group of spacer groups and therapeutic agent be cytotoxic chemical functional group's phase bonding for example, and other chemical functional group of spacer groups is for the linking group phase bonding of the chemical functional group with targeting agent or cleavable.The chemical functional group's of spacer groups example comprises hydroxyl, sulfydryl, carbonyl, carboxyl, amino, ketone group and sulfydryl.

Use L ¹the self sacrifice type linking group representing normally replaces or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl or replacement or unsubstituted assorted alkyl.In one embodiment, alkyl or aryl can comprise 1 to 20 carbon atom.They also can comprise polyalkylene glycol moiety.

Exemplary spacer groups for example comprises, 6-amino-hexanol, 6-sulfydryl hexanol, 10-hydroxydecanoic acid, glycine and other aminoacid, 1,6-hexanediol, Beta-alanine, 2-ethylaminoethanol, Phthalide, carbonyl, aminal ester, nucleic acid and the peptide etc. of mercaptamine (2-aminoethane mercaptan), 5-aminovaleric acid, 6-aminocaprolc acid, 3-maleimide benzoic acid, Phthalide, alpha-substituted.

Spacer groups can be for introducing other molecular weight and chemical functional group in cytotoxin-targeting agent complex.Usually, other quality and functional group can affect serum half-life and other character of complex.Therefore,, by careful selection spacer groups, can generate the cytotoxin complex with certain limit serum half-life.

Directly the spacer groups adjacent with drug moiety is also expressed as (L ¹) _m, wherein, m is selected from 0,1,2,3,4,5 or 6 integer.When there being a plurality of L ¹during spacer groups, can use identical or different spacer groups.L ¹can be any self sacrifice type group.In one embodiment, L ¹the assorted alkyl of the alkyl preferably replacing, unsubstituted alkyl, replacement and unsubstituted assorted alkyl, unsubstituted Heterocyclylalkyl and the Heterocyclylalkyl replacing.When medicine-ligand conjugates comprises hydrazine linking group, L ¹not containing disulphide bond.

L ⁴utilize the linking group that comprises this part that conjugate dissolubility is increased or assemble the linking group part that character reduces.L ⁴linking group is self sacrifice type not necessarily.In one embodiment, L ⁴part is assorted alkyl or the unsubstituted assorted alkyl of the alkyl replacing, unsubstituted alkyl, the aryl of replacement, unsubstituted aryl, replacement, and any one in them can be straight chain, side chain or ring-type.Substituent group for example can be, rudimentary (C ¹-C ⁶) alkyl, alkoxyl, alkylthio group, alkylamino or dialkylamino.In certain embodiments, L ⁴comprise non-annularity part.In another embodiment, L ⁴the amino acid polymer that comprises any lotus positive electricity or bear electricity, for example polylysine or poly arginine.L ⁴can comprise polymer, for example polyalkylene glycol moiety.In addition, L ⁴linking group comprises for example component of polymer and slightly chemical part.

In preferred embodiments, L ⁴comprise Polyethylene Glycol (PEG) part.L ⁴peg moiety can be 1 Dao50Ge unit head.Preferably, PEG has 1-12 recurring unit, more preferably, and 3-12 recurring unit, more preferably, 2-6Ge recurring unit, or even more preferably, 3-5Ge recurring unit, most preferably 4 recurring units.L ⁴can be formed by independent peg moiety, or it also can comprise other replacement or unsubstituted alkyl or assorted alkyl.In conjunction with PEG as L ⁴a part for part is useful for the water solublity that improves complex.In addition, peg moiety has reduced the concentration class that may occur in medicine and antibodies process.

(1) peptide linking group (F)

As discussed above, peptidyl linking group of the present invention can represent with following formula: (L ⁴) _p-F-(L ¹) _m, wherein, the linking group part that F representative comprises peptide base section.In one embodiment, F partly comprises optional other self sacrifice type linking group L ², and carbonyl.In another embodiment, F partly comprises amino and optional spacer groups L ³.

Correspondingly, in one embodiment, the conjugate that comprises peptidyl linking group comprises formula 4 structures:

In this embodiment, L ¹self sacrifice type linking group as above, and L ⁴make as mentioned above dissolubility increase or assemble the part that character reduces.L ²represent self sacrifice type linking group.M is 0,1,2,3,4,5 or 6; O and p are 0 or 1 independently.In one embodiment, m is 3,4,5 or 6.AA ¹represent one or more natural amino acids and/or non-natural a-amino acid; C is 1 to 20 integer.

In the peptide linking group of the present invention of above formula 4, AA ¹at its amino terminal, be directly connected to L ⁴, or work as L ⁴while not existing, be directly connected to X ⁴group (being targeting agent, detectable labelling, shielded reactive functional groups or not protected reactive functional groups).In some embodiments, when there being L ⁴time, L ⁴do not comprise and (AA ¹) _cthe direct connected carboxylic acyl radical of N-end.Thereby, in these embodiments, at L ⁴or X ⁴and AA ¹between directly exist carboxylic acyl radical unit optional, and this is at United States Patent (USP) the 6th, 214, necessary in the peptide linking group of No. 345.

In another embodiment, the conjugate that comprises peptidyl linking group comprises formula 5 structures:

In this embodiment, L ⁴it is the part that increases as mentioned above dissolubility or reduce to assemble character; L ³the spacer groups that comprises primary amine or secondary amine or carboxyl functional group, L ³amine and the pendant carboxy group functional group of D form amido link, or L ³carboxyl and the suspension amine functional group of D form amido link; With o and p be 0 or 1 independently.AA ¹represent one or more natural amino acids and/or non-natural a-amino acid; C is the integer between 1 to 20.In this embodiment, there is not L ¹(being that m is 0 in general formula).

In the peptide linking group of the present invention with above formula 5, AA ¹it amino terminal directly and L ⁴be connected, or work as L ⁴while not existing, direct and X ⁴group be connected (being targeting agent, detectable labelling, shielded reactive functional groups or not protected reactive functional groups).In some embodiments, when there being L ⁴time, L ⁴do not comprise direct and (AA ¹) _cthe connected carboxylic acyl radical of N-end.Therefore, in these embodiments, at L ⁴or X ⁴and AA ¹between directly to have carboxylic acyl radical unit be not essential, and this is at United States Patent (USP) the 6th, 214, necessary in the peptide linking group of No. 345.

Self sacrifice type linking group L ²

Self sacrifice type linking group L ²be difunctional chemical part, it can form normally stable three part molecules by the chemical part at two intervals is covalently bound together, and the mode by enzymatic lysis discharges the chemical part at described interval from three part molecules; And after described enzymatic lysis, while cracking from the remainder of molecule discharges the other parts of the chemical part at described interval.According to the present invention, self sacrifice type spacer groups is connected with peptide moiety covalency at an one end, and be connected with the chemical reaction position covalency of drug moiety at another end, the derivatization of this drug moiety has suppressed pharmacological activity, with peptide moiety is spaced apart with drug moiety and covalently boundly become three part molecules, in the situation that not there is not target enzyme, this three parts molecule is stable and pharmacology's non-activity, but by this target enzyme, place at the key of covalently bound spacer groups part and peptide moiety, this three parts molecule is enzyme cleavable, thereby affect peptide moiety discharges from three part molecules.This enzymatic lysis product will activate the self sacrifice characteristic of spacer groups part successively, and starts the spontaneous cracking of key that covalently bound spacer groups partly arrives drug moiety, thereby affects the release of the medicine of pharmacological activity form.

Self sacrifice type linking group L ²can be any self sacrifice type group.Preferably, L ²heterocyclylalkyl, replacement and unsubstituted aryl and replacement and the unsubstituted heteroaryl of the alkyl replacing, unsubstituted alkyl, the assorted alkyl of replacement, unsubstituted assorted alkyl, unsubstituted Heterocyclylalkyl, replacement.

A particularly preferred self sacrifice type spacer groups L ²available formula 6 represents:

The aromatic ring of ammonia benzyl can be replaced by one or more " K " group." K " group is the substituent group on aromatic ring, and it has replaced and the other hydrogen being connected belonging in four unsubstituted carbon of a part of ring structure." K " group can be single atom, halogen for example, or can be polyatom group, for example alkyl, assorted alkyl, amino, nitro, hydroxyl, alkoxyl, haloalkyl and cyano group.Each K is independently selected from the alkyl replacing, unsubstituted alkyl, the assorted alkyl of replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ²¹r ²², NR ²¹cOR ²², OCONR ²¹r ²², OCOR ²¹and OR ²¹, wherein, R ²¹and R ²²heterocyclylalkyl and unsubstituted Heterocyclylalkyl independently selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, replacement.Exemplary K substituent group includes but not limited to F, Cl, Br, I, NO ₂, OH, OCH ₃, NHCOCH ₃, N (CH ₃) ₂, NHCOCF ₃and methyl.For " Ka ", a is 0,1,2,3 or 4 integer.In a preferred embodiment, a is 0.

The ether oxygen atom of above structure is connected with carbonyl.From NR ²⁴functional group to the line in aromatic ring show amine functional group can with 5 carbon atoms in any one phase bonding, 5 carbon atoms had not only formed ring but also not by-CH ₂-O-group replaces.Preferably, the NR of X ²⁴functional group is with respect to-CH ₂the para-position of-O-group is connected with aromatic ring covalency.R ²⁴be selected from assorted alkyl and the unsubstituted assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement.In specific embodiments, R ²⁴hydrogen.

In preferred embodiments, the invention provides the peptide linking group of above formula (4), wherein, F comprises following structure:

Wherein,

A is 0,1,2,3 or 4 integer.

In another embodiment, the peptide linking group of above formula (4) comprise comprise following structure-F-(L ¹) _m-:

Wherein,

Spacer groups L ³

Spacer groups L ³be characterised in that it comprises primary amine or secondary amine or carboxyl functional group, or L ³the pendant carboxy group functional group of the amine of group and D forms amido link, or L ³carboxyl and the suspension amine functional group of D form amido link.L ³can be selected from and replace or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl or replacement or unsubstituted Heterocyclylalkyl.In preferred embodiments, L ³comprise aromatic group.More preferably, L ³comprise benzoic acid group, aniline group or indolyl radical.Can be used as-L ³the limiting examples of-NH-spacer groups comprises following structure:

Wherein, Z is selected from O, S and NR ²³, and

By comprising L ³the cracking of linking group of the present invention, L ³part keeps being connected with medicine D.Correspondingly, select L ³part, its existence that makes to be connected with D can significantly not change the activity of D.In another embodiment, medicine D part itself plays L ³the effect of spacer groups.For example, in one embodiment, medicine D is duocarmycin SA derivant, and wherein, drug moiety plays L ³the effect of spacer groups.The limiting examples of this embodiment comprises, wherein NH ₂-(L ³)-D has and is selected from which of following structure:

With

Wherein, Z is selected from O, S and NR ²³,

Wherein, R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; With

Wherein, each structural NH ₂group and (AA ¹) _creaction formation-(AA ¹) _c-NH-.

Peptide sequence AA ¹

Group AA ¹represent single amino acids or a plurality of aminoacid that link together by amido link.Aminoacid can be natural amino acid and/or non-natural a-amino acid.

Peptide sequence (AA ¹) _cit in function, is (amidification) residue that single amino acids (when c=1) or a plurality of amino acid whose amidatioon that links together by amido link are modified.Select peptide of the present invention, to pass through the enzyme pacemaker enzyme catalytic pyrolysis peptide of region of interest in biosystem.For example, for use targeting agent concerning cell-targeting then by the conjugate of Cell uptake, select by the peptide of one or more lysosomal protein enzymatic lysises, make peptide in lysosome by cracking in born of the same parents.Amino acid number in peptide can from 1 to 20; But more preferably, will have 2-8 aminoacid, a 2-6 aminoacid or 2,3 or 4 aminoacid, these aminoacid all comprise (AA ¹) _c.Being easy to by the peptide sequence of specific enzyme or enzyme cracking is that prior art is known.

A lot of peptide sequences by the enzymatic lysis in serum, liver, intestinal etc. are that prior art is known.The exemplary peptide sequence of the present invention comprises by the peptide sequence of protease cracking.After continuing after a while to use the sequence of protease-sensitivity, the focus of discussion is the clarification of explaining, is not limited to scope of the present invention.

When the enzyme of cleavage of peptide is protease, linking group generally includes the peptide of the protease recognition sequence that comprises cleavable.The protease recognition sequence of cleavable is the specific aminoacid sequence of being identified by protease in proteolytic cleavage process.A lot of protease cuttings site is that prior art is known, and these and other cleavage site can be included in linking group part.Referring to such as people Science 247:954 (1990) such as Matayoshi; The people Meth.Enzymol.241:254 (1994) such as Dunn; The people Meth.Enzymol.244:175 (1994) such as Seidah; Thornberry, Meth.Enzymol.244:615 (1994); The people Meth.Enzymol.244:595 (1994) such as Weber; The people Meth.Enzymol.244:412 (1994) such as Smith; The people Meth.Enzymol.248:614 (1995) such as Bouvier; The people such as Hardy, in AMYLOID PROTEIN PRECURSOR IN DEVELOPMENT, AGING, ANDALZHEIMER ' SDI SEASE, the people such as ed.Masters, 190-198 page (1994).

According to it by specific molecular for example the fitness of the protease selectivity enzymatic lysis of Tumor-assaciated select peptide sequence (AA ¹) _caminoacid.Aminoacid used can be natural or alpha-non-natural amino acid.They can be L or D configuration.In one embodiment, use at least three different aminoacid.In another embodiment, only use two aminoacid.

In preferred embodiments, according to it, by the ability of lysosomal protein enzymatic lysis, selected peptide sequence (AA ¹) _c, its limiting examples comprises cathepsin B, C, D, H, L and S.Preferably, peptide sequence (AA ¹) _ccan be organized in vitro Cathepsin B cracking, they can be tested by the known external protease cracking algoscopy of prior art.

In another embodiment, according to it, by the ability of the protease cracking of Tumor-assaciated, selected peptide sequence (AA ¹) _c, the protease of Tumor-assaciated is wherein for example the protease that near extracellular tumor cell is found, its limiting examples comprises thimet oligopeptidase (TOP) and CD10.Peptide can be tested by the known external protease cracking algoscopy of prior art by the ability of TOP or CD10 cracking.

The suitable limiting examples that is applicable to the peptide sequence of conjugate of the present invention comprises Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N ⁹-tosyl-Arg, Phe-N ⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO:1), β-Ala-Leu-Ala-Leu (SEQ ID NO:2) and Gly-Phe-Leu-Gly (SEQ ID NO:3).Preferred peptide sequence is Val-Cit and Val-Lys.

In another embodiment, from lower group, select to be positioned near aminoacid drug moiety: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.In other embodiments, from lower group, select to be positioned near aminoacid drug moiety: Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

Protease has participated in cancer metastasis.The synthetic increase of protease urokinase improves relevant with transfer ability in a lot of cancers.Urokinase activates into trypsinlike enzyme tryptogen, and tryptogen is arranged in ECS ubiquitously, and its activation can cause the degraded of extracellular matrix protein, and by this process, metastatic tumour cell is invaded.Trypsinlike enzyme also can activate collagenase, thereby promotes blood capillary and the lymphsystem collagen degradation in basement membrane around, thereby makes tumor cell invade in target tissue people Adv.Cancer.Res. such as (, 44:139 (1985)) Dano.Therefore, utilization can be by the peptide sequence of urokinase cracking as linking group also within the scope of the invention.

The present invention also provides the purposes to the peptide sequence of trypsinlike enzyme cracking sensitivity.The mast cell-expressed at least four kinds of different trypsinlike enzymes of people, are called α β I, β II and β III.These enzymes are not subject to the control of plasma protein enzyme inhibitor, and external only cracking minority physiology substrate.The trypsinlike enzyme family of serine protease has participated in various allergia and the inflammatory diseasess that involve mastocyte, because find that in suffering from patient's biological fluid of these obstacles trypsinlike enzyme level raises.But, trypsinlike enzyme in disease pathophysiology really corner cut color still need to be described.The scope of the biological function of trypsinlike enzyme and corresponding physiology's consequence is defined by their substrate specificity substantially.

Trypsinlike enzyme is the strong activator of pro-urokinase tryptogen activator (uPA), i.e. the zymogen forms of the protease relevant with invasion and attack with neoplasm metastasis.The activation of tryptogen cascade causes blending for extracellular the destruction of the extracellular matrix of dividing a word with a hyphen at the end of a line, this activation may be that pro-urokinase tryptogen activator is at the function (people such as Stack, Journalof Biological Chemistry 269 (13): 9416-9419 (1994)) of the P4-P1 of Pro-Arg-Phe-Lys sequence (SEQ ID NO:4) trypsinlike enzyme activation.Vasoactive intestinal peptide is the adjusting that neuropeptide participates in blood vessel permeability, also by trypsinlike enzyme cracking, mainly at Thr-Arg-Leu-Arg (SEQ ID NO:5) sequence (people such as Tam, Am.J.Respir.Cell Mol.Biol.3:27-32 (1990)).With the receptor PAR-2 of G albumen coupling can be in Ser-Lys-Gly-Arg (SEQ ID NO:6) sequence by trypsinlike enzyme cracking and activation, be driven into fibrocellular propagation, and by the receptor PAR-1 of thrombin activation in Pro-Asn-Asp-Lys (SEQ ID NO:7) sequence by trypsinlike enzyme deactivation (Molino etc., Journal of Biological Chemistry 272 (7): 4043-4049 (1997)).In a word, this evidence has pointed out trypsinlike enzyme the Zhong of the tissue remodelling center role as disease consequence.This is consistent with viewed profound change in the obstacle of some mast cell mediated.Chronic asthma and other long-term respiratory disorder a kind of proves fibre modification and pathological tissues thickens, and this may be the result of trypsinlike enzyme to the activation of its physiology target.Similarly, a series of reports show, in various cancers, angiogenesis and Mast Cell Density, trypsinlike enzyme activity relevant with prognosis mala (people such as Coussens, Genes and Development13 (11): 1382-97 (1999)); The people such as Takanami, Cancer 88 (12): 2686-92 (2000); The people such as Toth-Jakatics, Human Pathology 31 (8): 955-960 (2000); The people such as Ribatti, International Journal of Cancer85 (2): 171-5 (2000)).

For evaluate specific protease whether the method for the selected peptide sequence of cracking be known in the art.For example, the use of 7-amino-4-methylcoumarin (AMC) fluorescence peptide substrates is the maturation method (Zimmerman, the people such as M., (1977) Analytical Biochemistry 78:47-51) about protease specific assay.The specificity cracking of N-anilide key discharges fluorescence AMC leaving group, allows to measure simply the heating rate of single substrate.Recently, by get sample widely in single experiment, adopted the array (Lee in AMC peptide substrates library, D. wait people, Bioorganic and Medicinal Chemistry Letters9:1667-72) and scanning library, position (Rano (1999), T.A. wait people, (1997) Chemistry andBiology 4:149-55) describe fast the N-end specificity of protease.Thereby those skilled in the art can easily evaluate one group of peptide sequence, to determine their practicality in the present invention, without appealing to too much experimental technique.

(2) hydrazine linking group (H)

In second embodiment, conjugate of the present invention comprises hydrazine self sacrifice type linking group, and wherein, conjugate has following structure:

Wherein, D, L ¹, L ⁴and X ⁴as above definition, and further describe in this article, and H is the linking group that comprises following structure:

Wherein,

N ₁it is the integer of 1-10;

N ₂0,1 or 2;

I or key (being the key between main chain carbon and adjacent nitrogen), or

Wherein, n ₃be 0 or 1, condition is to work as n ₃0 o'clock, n ₂not 0; With

N ₄1,2 or 3,

Wherein, when I is key, n ₁3 and n ₂that 1, D can not be

Wherein, R is Me or CH ₂-CH ₂-NMe ₂.

In one embodiment, the replacement on benzyl ring is para-orientation.In preferred embodiments, n ₁2,3 or 4, or n ₁3.In preferred embodiments, n ₂1.In preferred embodiments, I is key (being the key between main chain carbon and adjacent nitrogen).In one aspect, hydrazine linking group, H, can form 6-unit self sacrifice type linking group by cracking, for example, works as n ₃0 and n ₄it is 2 o'clock.In one aspect of the method, hydrazine linking group, H, can form two 5-unit self sacrifice type linking groups by cracking.In other side, by cracking, H forms 5-unit self sacrifice type linking group, and H forms 7-unit self sacrifice type linking group, or H forms 5-unit's self sacrifice type linking group and 6-unit self sacrifice type linking group.Rate of cleavage is affected by the size that forms ring by cracking.Therefore,, according to required rate of cleavage, can select the suitably large circlet forming by cracking.

5 yuan of hydrazine linking groups

In one embodiment, hydrazine linking group comprises 5-unit hydrazine linking group, and wherein, H comprises following structure:

In preferred embodiments, n ₁2,3 or 4.In a further preferred embodiment, n ₁3.In above structure, each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement.In one embodiment, each R ²⁴h or C independently ₁-C ₆alkyl.In another embodiment, each R ²⁴h or C independently ₁-C ₃alkyl more preferably, is H or CH ₃.In another embodiment, at least one R ²⁴it is methyl.In another embodiment, each R ²⁴h.Select each R ²⁴steric effect and change dissolubility with complex compounds.

5-unit hydrazine linking group can carry out the one or more cyclizations from linking group separate drug, and can use-case as shown in the formula describing:

The exemplary synthetic route of 5 yuan of linking groups of the present invention is:

The DMDA b of Cbz-protection and 2,2-dimethyl-malonic acid a react and form Cbz-DMDA-2,2-dimethyl malonic acid c in thionyl chloride solution.Compound c reacts and forms DMDA-2,2-dimethyl malonic acid-Boc-N-methyl hydrazine e with Boc-N-methyl hydrazine d under the existence of hydrogen.

hexa-atomic hydrazine linking group

In another embodiment, hydrazine linking group comprises 6-unit hydrazine linking group, and wherein, H comprises following structure:

In preferred embodiments, n ₁3.In above structure, each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement.In one embodiment, each R ²⁴h or C independently ₁-C ₆alkyl.In another embodiment, each R ²⁴h or C independently ₁-C ₃alkyl, more preferably, H or CH ₃.In another embodiment, at least one R ²⁴it is methyl.In another embodiment, each R ²⁴h.Select each R ²⁴steric effect and change dissolubility with complex compounds.In preferred embodiments, H comprises following structure:

In one embodiment, H comprises two dimethyl replacements.In an embodiment of above structure, each R ²⁴h or replacement or unsubstituted alkyl independently.

6-unit hydrazine linking group is the cyclization from linking group separate drug by experience, and can use-case as shown in the formula describing:

The exemplary synthetic route of preparing 6 yuan of linking groups of the present invention is:

The dimethyl propylene propylhomoserin a of Cbz-protection reacts the dimethyl propylene aminoacylhydrazine b that forms Cbz-protection in dichloromethane solution with HOAt and CPI.Hydrazine b, by the protection that spends of methanol, forms compound c.

other hydrazine linking group

Pay close attention to the present invention and comprise 7 yuan of linking groups.This linking group can not resemble 5 yuan or 6 yuan of linking groups ring formation so fast, but this linking group is preferred for some drugs-ligand conjugates.Similarly, hydrazine linking group can comprise two 6 rings or have the hydrazine linking group of one 6 yuan and one 5 ring products.Also pay close attention to 5 yuan and 7 yuan of linking groups and 6 yuan and 7 yuan of linking groups.

Other hydrazine structure, H, has following formula structure:

Wherein, q is 0,1,2,3,4,5 or 6; With

Each R ²⁴assorted alkyl and unsubstituted assorted alkyl independently selected from the alkyl of H, replacement, unsubstituted alkyl, replacement.This hydrazine structure also can form 5 yuan, 6 yuan or 7 rings, and can add other component to form polynary ring.

(3) disulphide linking group (J)

In another embodiment, the disulphide group that linking group comprises enzyme cleavable.In one embodiment, the invention provides the cytotoxic drug-ligand compound with formula 3 structures:

X ⁴

L ⁴ _pJ

L ¹ _m

D (3)

Wherein, D, L ¹, L ⁴and X ⁴as above definition also further describes in this article, and J is the disulphide linking group that comprises following building stone:

Wherein,

A is 0,1,2,3 or 4 integer; With

D is 0,1,2,3,4,5 or 6 integer.

The aromatic ring of disulphide linking group can be replaced by one or more " K " group." K " group is the substituent group on aromatic ring, and it has replaced the hydrogen of one being connected in addition in four non-substituted carbon, these four parts that non-substituted carbon is ring structure." K " group can be single atom, halogen for example, or can be polyatom group, for example alkyl, assorted alkyl, amino, nitro, hydroxyl, alkoxyl, haloalkyl and cyano group.Exemplary K substituent group includes but not limited to F, Cl, Br, I, NO ₂, OH, OCH ₃, NHCOCH ₃, N (CH ₃) ₂, NHCOCF ₃and methyl.For " Ka ", a is 0,1,2,3 or 4 integer.In a specific embodiments, a is 0.

In preferred embodiments, linking group comprises the disulphide group of the enzyme cleavable of following formula:

In this embodiment, L ⁴, X ⁴, p and R ²⁴discriminating as mentioned above, and d is 0,1,2,3,4,5 or 6.In specific embodiments, d is 1 or 2.

Disulphide linking group is presented in following formula more specifically:

The instantiation of this embodiment is as follows:

Preferably, d is 1 or 2.

Another disulphide linking group is presented in following formula:

The instantiation of this embodiment is as follows:

Preferably, d is 1 or 2.

In different embodiments, disulphide and amine ortho position.In another embodiment, a is 0.In preferred embodiments, R ²⁴independently selected from H and CH ₃.

The exemplary synthetic route of preparing disulphide linking group of the present invention is as follows:

3-mercaptopropionic acid a solution reacts with aldrithiol-2 and forms 3-methylbenzothiazole iodide (3-methyl benzothiazolium iodide) b.3-methylbenzothiazole iodide c reacts with sodium hydroxide and forms compound d.The methanol solution of compound d further reacts with compound b and forms Verbindung.Verbindung is formed compound f by the effect deprotection of chloroacetic chloride and methanol.

Medicine-ligand conjugates of the present invention can optionally comprise two or more linking groups.These linking groups can be identical or different.For example, peptidyl linking group can be used for medicine to be connected to part, and second peptidyl linking group can connection diagnostic agent arrive complex.Alternatively, any one peptidyl, hydrazine or disulphide linking group can connect medicine and ligand complex, and any one peptidyl, hydrazine or disulphide linking group can connection diagnostic agent arrive complex.Other application of other linking group comprises that linking parsing agent, biomolecule, targeting agent and detectable labelling are to medicine-ligand complex.

The compounds of this invention of multivalence also within the scope of the invention, comprises dimer, trimer, the tetramer and the higher homologue of the compounds of this invention for example or its reactive analog.Polyad can from single or more than one the compounds of this invention assemble and to form.For example, dimerization construct can be " all dimerization " or " assorted dimerization ".And the multivalence construct that wherein the compounds of this invention or its reactive analog are connected with oligomeric or aggregation framework (such as polylysine, glucosan, hetastarch etc.) also within the scope of the invention.Framework is multi-functional (for example having for connecting a group reaction position of the compounds of this invention) preferably.And framework can utilize single or an above the compounds of this invention is derivative.

And, the present invention includes such compound, after they functionalised, the water solublity of gained compound is with respect to not strengthened by similar functionalized analogue compounds.Thereby any substituent group as herein described can be replaced by the stronger similar atomic group of water solublity.For example, within the scope of the present invention, with glycol, replace hydroxyl, or replace amine by the higher similar part of quaternary amine, azanol or water solublity.In preferred embodiments, for compound ions passage described herein, not that active essential position replaces with strengthening the water miscible part of parent compound, give extra water solublity.It is known in the art strengthening the water miscible method of organic compound.These class methods include but not limited to organic core functionalized by permanent charged part, quaternary ammonium for example, or be used in charged group functionalization under the relevant pH of physiology, for example carboxylic acid, amine.Additive method comprises to the additional group that contains hydroxyl or amine of organic core, such as alcohol, polyhydric alcohol, polyethers etc.Representative example includes but not limited to polylysine, polymine, PEG and gathers (propylene glycol).The functionalized chemistry and the strategy that are suitable for these compounds are known in the art.For example, referring to Dunn, R.L., waits people, Eds.POLYMERIC DRUGS AND DRUG DELIVERYSYSTEMS, ACS Symposium Series Vol.469, American ChemicalSociety, Washington, D.C.1991.

medicine

Medicine, is described as " D " in this article, is provided as in the present invention a part for medicine-ligand conjugates, and wherein, medicine is connected to part by peptidyl, hydrazine or disulphide linking group.Medicine must have the biologic activity of wanting, and comprises reactive functional groups to be connected with part.The biologic activity of wanting comprises diagnosis, cures, alleviates, treats or prevents animal as people's disease.Therefore,, as long as it has required reactive functional groups, term " medicine " refers to approved chemical drugs in legal American Pharmacopeia, legal Homeopathic Pharmacopeia of the United States or legal NF or its any supplementary issue.Exemplary medicine is set forth in Physician ' s Desk Reference (PDR) and in the orange paper of U.S. food and Drug Administration (FDA) maintenance.New drug is found and develops continuing, and the invention provides these new drugs and also can mix in medicine-ligand complex of the present invention.

Preferred functional group comprises primary amine or secondary amine, hydroxyl, sulfydryl, carboxyl, aldehyde and ketone.Preferred functional group comprises hydroxyl, primary amine or secondary amine, sulfydryl and carboxylic acid functional.Even preferred functional group comprises hydroxyl, primary amine and secondary amine and carboxylic acid functional.Medicine must have at least one, but can have 2,3,4,5,6 or a plurality of reactive functional groups.In addition, self sacrifice type spacer groups, L ¹, can be incorporated between the reactive functional groups and peptide, hydrazine or disulphide linking group of medicine.

Medicine-ligand conjugates is effective for common object, in this object, corresponding medicine is effectively, but due to the intrinsic ability (being useful especially at this cell Chinese medicine) in expectation cell that medicine is transferred to of part, corresponding medicine has more superior effect.

Exemplary medicine comprises protein, peptide and the small-molecule drug that comprises the functional group that is connected to part.More particularly, these medicines for example comprise, enzyme inhibitor, for example dihydrofolate reductase inhibitor and thymidylic acid synthase inhibitor, DNA intercalator, DNA cutting agent, topoisomerase enzyme inhibitor, anthracene nucleus medicament family, Vinca medicine, mitomycin, bleomycin, cytotoxin nucleoside, pteridine medicine family, diynenes, podophyllotoxin, differentiating inducer and taxol.

The preferred medicine of the present invention comprises the cytotoxic drug and other micromolecule, protein or the polypeptide with required biologic activity, for example toxin for treatment of cancer.Can select medicine to activate by being attached to tumor-targeting ligands in tumor cell.These tumour-specific medicine-ligand conjugates have the tumour-specific bringing due to ligand specificity.The example is following medicine-ligand conjugates, and it is the high selectivity substrate of tumour-specific enzyme, and wherein, these enzymes are present near tumor with the amount near the free drug of cellulation toxic level tumor of being enough to.An advantage of these tumour-specific medicine-ligand complex is that they are stable to protease accidental in human serum.Another advantage of medicine-ligand complex is that they have lower toxicity than corresponding free drug; In addition, the specificity of complex can allow to use the systemic concentrations lower with respect to free drug, because specificity raising can cause the complex of higher percentage ratio, is present in tumor locus.

cytotoxin

For cytotoxic drug of the present invention, for example comprise, duocarmycin SA and CC-1065 and analog thereof, comprise duocarmycin SA and CC-1065 with CBI (1, 2, 9, 9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone) be basic analog, with MCBI (7-methoxyl group-1, 2, 9, 9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone) be basic analog, with with CCBI (7-cyano group-1, 2, 9, 9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone) be basic analog, doxorubicin and doxorubicin conjugate, for example morpholino-doxorubicin and cyano group morpholino-doxorubicin, dolastatin, dolastatin-10 for example, combretastatin, calicheamycin, maitansine, maitansine analog, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoyl valeric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, paclitaxel, Docetaxel, SN-38, hycamtin, rhizomycin, Quinomycin A., colchicine, vinblastine, vindesine, estramustine, Cemadotin, Eleutherobin., methotrexate, methopterin, dichioromethotrexate, 5-fluorouracil, 6-MP, cytosine arabinoside, melphalan, leurosine, inrosidine, D actinomycin D, daunorubicin and daunorubicin conjugate, ametycin, Mitomycin A, carubicin, aminopterin, talisomycin, podophyllotoxin and podophyllotoxin derivative, for example etoposide or etoposide phosphate, vincristine, taxol, taxotere tretinoin, butanoic acid, N ⁸-acetyl spermidine, camptothecine and analog thereof.Can modify other known medicine, with the functional group being provided for linking group described herein is combined.Such chemical modification is known in the art.

Being preferred for cytotoxin of the present invention comprises: duocarmycin SA and CC-1065 with and take that CCBI is basis and take MCBI as basic analog, morpholino-doxorubicin, cyano group morpholino-doxorubicin, dolastatin-10, combretastatin, calicheamycin, maitansine, DM-1, auristatin E, AEB, AEFP, MMAE, Tubulysin A, Disorazole, EPOTHILONE A and EPO906.

The particularly preferred cytotoxin of the present invention is effective duocarmycin SA derivant and CC-1065.Parent compound is special effective antitumour antibiotic, and it brings into play its biological action (people J.Org.Chem.55:4499 (1990) such as Boger by the reversible DNA sequence selectivity alkanisation that controlled by stereo selectivity; The people J.Am.Chem.Soc.112:8961 (1990) such as Boge r; The people such as Boger, J.Am.Chem.Soc.113:6645 (1991); The people J.Am.Chem.Soc.115:9872 (1993) such as Boger; The people such as Boger, Bioorg.Med.Chem.Lett.2:759 (1992)).After duocarmycin SA is open at first, the selection of DNA alkanisation and the structure origin thereof of much making great efforts to be devoted to illustrate duocarmycin SA have been done.

The particularly preferred aspect of the present invention provides the cytotoxic compound with formula 7 structures:

Wherein ring system A is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl.Exemplary ring system comprises phenyl and pyrroles.

Symbol E and G are independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, hetero atom, singly-bound, or E and G be optionally connected to form ring system, this ring system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl.

Symbol X is selected from O, S and NR ²³.R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.

Symbol R ³be selected from (=O), SR ¹¹, NHR ¹¹and OR ¹¹, wherein, R ¹¹be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, bisphosphate, triguaiacyl phosphate, acyl group, C (O) R ¹²r ¹³, C (O) OR ¹², C (O) NR ¹²r ¹³, P (O) (OR ¹²) ₂, C (O) CHR ¹²r ¹³, SR ¹²or SiR ¹²r ¹³r ¹⁴.Symbol R ¹², R ¹³and R ¹⁴represent independently H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein, R ¹²and R ¹³together with the nitrogen-atoms connecting with them or carbon atom, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally contains two or more hetero atoms.R ¹², R ¹³and R ¹⁴in one or more cleavable groups that can comprise in its structure.

R ⁴, R ⁴', R ⁵and R ⁵' independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, SR ¹⁵, OR ¹⁵, CR ¹⁵=NR ¹⁶and O (CH ₂) _nn (CH ₃) ₂, wherein, n is 1 to 20 integer.R ¹⁵and R ¹⁶represent independently H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein, R ¹⁵and R ¹⁶together with the nitrogen-atoms connecting with them, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms.A demonstrative structure is aniline.

R ⁴, R ⁴', R ⁵, R ⁵', R ¹¹, R ¹², R ¹³, R ¹⁵and R ¹⁶optionally comprise the one or more cleavable groups in its structure.Exemplary cleavable group includes but not limited to peptide class, aminoacid, hydrazine and disulphide.

R ¹¹, R ¹², R ¹³, R ¹⁵and R ¹⁶in at least one for connecting medicine to linking group of the present invention, as described herein, be for example connected to L ¹, if present, or be connected to F, H or J.

In exemplary embodiment further, at least one R ⁴, R ⁴', R ⁵, R ⁵', R ¹¹, R ¹², R ¹³, R ¹⁵and R ¹⁶carry the reactive group that is suitable for puting together this compound.In further exemplary embodiment, R ⁴, R ⁴', R ⁵, R ⁵', R ¹¹, R ¹², R ¹³, R ¹⁵and R ¹⁶independently selected from the alkyl of H, replacement and the assorted alkyl of replacement, and there is reactive functional groups at the free-end of alkyl or assorted moieties.One or more R ⁴, R ⁴', R ⁵, R ⁵', R ¹¹, R ¹², R ¹³, R ¹⁵and R ¹⁶can put together with another kind of molecule such as targeting agent, detectable labelling, solid carrier etc.

From discussion herein, can obviously find out, work as R ¹⁵and R ¹⁶in at least one while comprising reactive functional groups, this functional group can be the component of the key between medicine and another kind of molecule.In exemplary embodiment, wherein, R ¹⁵and R ¹⁶in at least one comprise being connected between medicine and another kind of molecule, R ¹⁵and R ¹⁶in at least one by the part of enzymatic lysis.In further exemplary embodiment, R ⁴, R ⁴', R ⁵and R ⁵' at least one be:

In formula 8, symbol X ²and Z ¹representative is selected from O, S and NR independently ²³member.Radicals R ¹⁷and R ¹⁸independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁹r ²⁰, NC (O) R ¹⁹, OC (O) NR ¹⁹, OC (O) OR ¹⁹, C (O) R ¹⁹, SR ¹⁹or OR ¹⁹, condition is R ¹², R ¹³, R ¹⁹or R ²⁰in at least one comprise linking group of the present invention, as disclosed herein.

Symbol R ¹⁹and R ²⁰represent independently heteroaryl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted peptidyl, the wherein R of replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or replacement ¹⁹and R ²⁰together with the nitrogen-atoms connecting with them, optionally form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, optionally contain two or more hetero atoms, its condition is to work as Z ¹while being NH, R ¹⁷and R ¹⁸not H, and R ¹⁷not NH ₂.The symbol R that spreads all over this description ¹⁹and R ²⁰also contain R ⁴and R ⁵described group.Thereby for example, providing within the scope of the invention such compound, it has two or more as above described connected fused phenyl-heterocycle ring system or combinations of fused rings and linking group just now.And in there are those embodiments of linking group, linking group can be used as R ⁴, R ⁴', R ⁵or R ⁵' substituent group exists or as R ¹⁷or R ¹⁸substituent group exists.

R ⁶be singly-bound, it can exist also and can not exist.Work as R ⁶while existing, R ⁶and R ⁷connect and compose cyclopropyl rings.R ⁷cH ₂-X ¹or-CH ₂-.Work as R ⁷be-CH ₂in-time, it is the component of cyclopropane ring.Symbol X ¹represent leaving group, halogen for example, as Cl, Br or F.Technical staff will understand R ⁶and R ⁷compound mode can not violate valent principle.

This ring of curve representation in hexatomic ring can have one or more degrees of unsaturation, and can be armaticity.Thereby, such as the following scope that waits ring structure and relevant structure all to belong to formula (9):

with

In exemplary embodiment, ring system A replaces or unsubstituted benzyl ring.Ring system A is preferably replaced by the described aryl substituent of an one or more joint as defined herein.In a preferred embodiment, benzyl ring is partly replaced by CN or methoxyl group.

In another exemplary embodiment, the invention provides the compound with formula 10 structures:

In this embodiment, atomic group R ³, R ⁴, R ⁴', R ⁵, R ⁵', R ⁶, R ⁷with X be substituent group as above.Symbols Z is independently selected from O, S and NR ²³member.Symbol R ²³representative is selected from the member of H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.Each R ²³selected independently.Symbol R ¹represent H, replacement or unsubstituted low alkyl group or C (O) R ⁸or CO ₂r ⁸.R ⁸be selected from the alkyl of replacement, unsubstituted alkyl, NR ⁹r ¹⁰, NR ⁹nHR ¹⁰and OR ⁹.R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl.Atomic group R ²h or replacement or unsubstituted low alkyl group.Generally preferably work as R ²while being the alkyl replacing, it is not perfluoroalkyl, for example CF ₃.In one embodiment, R ²be the alkyl replacing, wherein, substituent group is not halogen.In another embodiment, R ²it is unsubstituted alkyl.

As discussed above, X ¹can be leaving group.Useful leaving group includes but not limited to that halogen, azide, sulphonic acid ester (such as alkyl sulphonyl, aryl sulfonyl), oxonium ion, alkyl perchloric acid ester, ammonium alkyl sulfonic acid ester, alkyl fluoride are for sulphonic acid ester and fluoric compound (such as triflate, nonaflates, tosylate) etc.Special halogen as leaving group is F, Cl and Br.Be suitable in the limit of power that is chosen in those skilled in the art of these and other leaving group of specific a set of reaction condition (for example, referring to March J, ADVANCED ORGANIC CHEMISTRY, the 2nd edition, John Wiley and Sons, 1992; Sandler SR, Karo W, the PREPARATIONS of ORGANIC functional group, the 2nd edition, AcademicPress, Inc., 1983; Wade LG, COMPENDIUM OF ORGANIC SYNTHETICMETHODS, John Wiley and Sons, 1980).

In exemplary embodiment, R ¹ester moiety, CO for example ₂cH ₃.In further exemplary embodiment, R ²be low alkyl group, it can be replacement or unsubstituted.At present preferred low alkyl group is CH ₃.In further embodiment, R ¹cO ₂cH ₃, and R ²cH ₃.

In another exemplary embodiment, R ⁴, R ⁴', R ⁵and R ⁵' independently selected from H, halogen, NH ₂, OMe, O (CH ₂) ₂n (Me) ₂and NO ₂.

In one embodiment, select medicine, so that leaving group X ¹be selected from halogen, alkyl sulphonyl, aryl sulfonyl and azide.In another embodiment, Z is O.In certain embodiments, R ¹can be CO ₂cH ₃or R ²can be CH ₃; In addition, R ¹can be CO ₂cH ₃and R ²can be CH ₃.R ⁴, R ⁴', R ⁵or R ⁵' in one can be C (O) R ¹⁵, and R ⁴, R ⁴', R ⁵and R ⁵' in other three be H.In addition, R ⁴, R ⁴', R ⁵and R ⁵' at least one can be to be different from and be selected from H and OCH ₃member.In one embodiment, R ⁴, R ⁴', R ⁵and R ⁵' independently selected from H, halogen, NH ₂, O (CH ₂) ₂n (Me) ₂and NO ₂.

In preferred embodiments, R ⁴, R ⁴', R ⁵or R ⁵' in one be O (CH ₂) ₂n (Me) ₂, and other R ⁴, R ⁴', R ⁵and R ⁵' be H.In another embodiment, R ⁷cH ₂-X ¹, wherein, X ¹f, Cl or Br, and R ⁶do not exist.

In another exemplary embodiment, the invention provides the compound with formula 11 and 12 structures:

with

In an embodiment of above formula, X is O preferably; With Z O preferably.In another embodiment, Z is NR ²³or O.Alternatively, R ⁴, R ⁴', R ⁵or R ⁵' in one can be O (CH ₂) ₂n (Me) ₂, and R ⁴, R ⁴', R ⁵or R ⁵' in other three be H.In one embodiment, R ⁴, R ⁴', R ⁵or R ⁵' can be selected from R ²⁹, COOR ²⁹, C (O) NR ²⁹, C (O) NNR ²⁹, wherein, R ²⁹be selected from heteroaryl and the unsubstituted heteroaryl of the assorted alkyl of ring of the assorted alkyl of the cycloalkyl of the alkyl of H, OH, replacement, unsubstituted alkyl, replacement, unsubstituted cycloalkyl, replacement, unsubstituted assorted alkyl, replacement, the assorted alkyl of unsubstituted ring, replacement.

In another embodiment of above formula, X is O preferably, and Z is O preferably, R ₁cO preferably ₂cH ₃, R ₇cH preferably ₂-Cl, R ₂cH preferably ₃, R ₃oH preferably.Alternatively, R ⁴, R ⁴', R ⁵or R ⁵' in one can be NHC (O) (C ₆h ₄) NH ₂, and R ⁴, R ⁴', R ⁵or R ⁵' in other three be H.

In one embodiment, R ²⁹can be selected from:

with

In another embodiment of medicine, be selected from R ⁴and R ⁵a member be:

Wherein, X ²and Z ¹independently selected from O, S and NR ²³; R ¹⁷and R ¹⁸independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁹r ²⁰, NC (O) R ¹⁹, OC (O) NR ¹⁹, OC (O) OR ¹⁹, C (O) R ¹⁹, OR ¹⁹and O (CH ₂) _nn (CH ₃) ₂.In this embodiment, n is 1 to 20 integer; R ¹⁹and R ²⁰independently selected from replacing or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl, wherein, R ¹⁹and R ²⁰together with the nitrogen-atoms connecting with them, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms, wherein, and R ¹¹, R ¹², R ¹³, R ¹⁵, R ¹⁶, R ¹⁹or R ²⁰in one connect described medicine to L ¹, if present, or be connected to F.In a preferred embodiment, X ²o and Z ¹o or NR ²³.

Another preferred structure of formula 7 duocarmycin SA analog is that wherein, ring system A is benzyl ring unsubstituted or that replace.For formula 7 structures when ring system A is pyrroles, the preferred substituents on drug molecule mentioned above is also the preferred substituents when ring system A is the benzyl ring that does not replace or replace.

For example, in preferred embodiments, medicine (D) comprises following structure:

In this structure, R ³, R ⁶, R ⁷, X is as described in above formula 7.In addition, Z is selected from O, S and NR ²³, wherein, R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group;

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸or CO ₂r ⁸, wherein, R ⁸be selected from NR ⁹r ¹⁰and OR ⁹, wherein, R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl;

R ¹' be H, replacement or unsubstituted low alkyl group or C (O) R ⁸, wherein, R ⁸be selected from NR ⁹r ¹⁰and OR ⁹, wherein, R ⁹and R ¹⁰independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl;

R ²h or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl or cyano group or alkoxyl; And R ²' be H or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl.

R ¹¹, R ¹², R ¹³, R ¹⁵or R ¹⁶in at least one connect medicine to L ¹, if present, or be connected to F, H or J.

In preferred embodiments, R ⁴, R ⁴', R ⁵or R ⁵' in one be O (CH ₂) ₂n (Me) ₂, R ⁴, R ⁴', R ⁵and R ⁵' in other several are H.In another embodiment, R ⁷cH ₂-X ¹, wherein, X ¹f, Cl or Br, and R ⁶do not exist.

In one embodiment, the invention provides the cytotoxic drug-ligand compound with following formula structure:

X ⁴ L ⁴

_pQ

L ¹

_m D ¹

Symbol L ⁴represent linking group part, and p is 0 or 1.L ⁴conjugate dissolubility is improved and assemble the part that character reduces.L ⁴the example of part comprises assorted alkyl or the unsubstituted assorted alkyl of the aryl of the alkyl of replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, any one in them can be straight chain, side chain or ring-type, the amino acid polymer of lotus positive electricity or bear electricity, for example polylysine or poly arginine or other polymer are as Polyethylene Glycol.

Symbol Q represents the linking group of any cleavable, includes but not limited to any peptidyl as herein described, hydrazone and disulphide linking group.Other suitable linking group includes but not limited to describe in Publication about Document which: United States Patent (USP) the 6th, 214, No. 345; No. the 2003/0096743rd, 2003/0130189 and 2004/121940, U.S. Patent Application Publication; No. 04/043493, PCT public announcement of a patent application WO 03/026577 and WO; With European patent application published EP1243276 and No. EP1370298, they all introduce text as a reference.The linking group of cleavable comprise can by chemistry or biological method selective splitting and by cracking from X ⁴isolate medicine D ¹which.Cracking can occur everywhere, according to the length of linking group or at any one end of linking group.

Symbol D ¹representative has the medicine of following structure:

Wherein, X and Z are independently selected from O, S and NR ²³;

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸or CO ₂r ⁸,

R ¹' be H, replacement or unsubstituted low alkyl group or C (O) R ⁸,

R ³be selected from SR ¹¹, NHR ¹¹and OR ¹¹, wherein, R ¹¹be selected from the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, bisphosphate, triguaiacyl phosphate, acyl group, C (O) R ¹²r ¹³, C (O) OR ¹², C (O) NR ¹²r ¹³, P (O) (OR ¹²) ₂, C (O) CHR ¹²r ¹³, SR ¹²and SiR ¹²r ¹³r ¹⁴, wherein, R ¹², R ¹³and R ¹⁴independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein, R ¹²and R ¹³together with the nitrogen connecting with them or carbon atom, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms;

R ⁷cH ₂-X ¹or in described cyclopropyl rings with R ⁶be connected-CH ₂-, wherein,

X ¹leaving group,

R ⁴, R ⁴', R ⁵and R ⁵' independently selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, SR ¹⁵, OR ¹⁵, CR ¹⁵=NR ¹⁶, and O (CH ₂) _nnR ²⁴r ²⁵wherein n is 1 to 20 integer;

R ¹⁵and R ¹⁶independently selected from H, replace or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein, R ¹⁵and R ¹⁶together with the nitrogen-atoms connecting with them, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms;

And R ²⁴and R ²⁵independently selected from unsubstituted alkyl, and wherein, R ⁴, R ⁴', R ⁵and R ⁵' at least one be O (CH ₂) _nnR ²⁴r ²⁵.

In some embodiments, n is 2.In some embodiments, R ²⁴and R ²⁵it is methyl.In some embodiments, R ⁴o (CH ₂) _nnR ²⁴r ²⁵, and R ⁴', R ⁵and R ⁵' be H.In some embodiments, R ⁴o (CH ₂) ₂n (CH ₃) ₂, and R ⁴', R ⁵and R ⁵' be H.In some embodiments, Q is the linking group that is selected from F as above, H and J.In some embodiments, R ¹, R ¹', R ²and R ²' be H.

Medicine D ¹preferred structure formula as follows:

Medicine D ¹another preferred structure formula as follows:

Medicine D ¹another preferred structure formula as follows:

With

In another exemplary embodiment of the present invention, cytotoxic drug can be tubulysin analog or related compound, for example, and the compound described in formula 13 structures:

Wherein, R ₁and R ₂h or low alkyl group, or more particularly isobutyl group, ethyl, propyl group or the tert-butyl group, and R ₃h or OH.Tubulysin and the purposes in treatment cancer thereof have for example been described in, in PCT communique WO 2004/005327 and WO 2004/005326.The production of tubulysin compound is described in DE10008089.Can be used for connecting tubulysin provides in an embodiment to the method for the various linking groups of the present invention.Preferred tubulysin analog is Tubulysin A-F.

cBI analog

These special compounds are CBI analog, are that they have mixed 1,2,9,9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone (CBI) alkanisation region or alkanisation subunit.This compound useful as drug.The preferred medicine of the present invention comprises the cytotoxic drug for treatment of cancer.These compounds may by or do not puted together, or comprise above-mentioned linking group.For cytotoxic drug of the present invention, for example comprise, with CBI (1,2,9,9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone) be basic analog, with MCBI (7-methoxyl group-1,2,9,9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone) be basic analog and with CCBI (7-cyano group-1,2,9,9a-tetrahydrochysene cyclopropane [c] benzo [e] indole-4-ketone) be basic analog.

In one embodiment, the compounds of this invention has following formula (14):

R ¹h, replacement or unsubstituted low alkyl group, C (O) R ⁸, CO ₂r ⁸,

R ¹' be H, replacement or unsubstituted low alkyl group or C (O) R ⁸,

R ⁶be singly-bound, it exists or does not exist, and when existing, R ⁶and R ⁷phase order even forms cyclopropyl rings; With

R ⁷cH ₂-X ¹, or in described cyclopropane basic ring with R ⁶be connected-CH ₂-, wherein, X ¹leaving group,

R ⁴, R ⁴', R ⁵and R ⁵' independently selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, SR ¹⁵, OR ¹⁵, CR ¹⁵=NR ¹⁶and O (CH ₂) _nnR ²⁴r ²⁵, wherein, n is 1 to 20 integer, preferably, n is 2 to 6 integer;

And R ²⁴and R ²⁵independently selected from unsubstituted alkyl, and

Wherein, R ⁴, R ⁴', R ⁵and R ⁵' at least one be O (CH ₂) _nnR ²⁴r ²⁵.

As discussed above, X ¹can be leaving group.Useful leaving group includes but not limited to that halogen, azide, sulfonic acid esters (such as alkyl sulphonyl, aryl sulfonyl), oxonium ion, perchloric acid Arrcostab, ammonium alkane sulfonic acid ester (ammonioalkanesulfonate esters), alkyl fluoride are for sulphonic acid ester and fluoride (such as triflates, nonaflates, tresylates) etc.Specific halogen as leaving group is F, Cl and Br.Within being suitable for the limit of power that is chosen in those skilled in the art of these and other leaving group of reaction condition of particular combination (referring to for example, March J, ADVANCED ORGANIC CHEMISTRY, the 2nd edition, JohnWiley and Sons, 1992; Sandler SR, Karo W, ORGANIC FUNCTIONALGROUP PREPARATIONS, the 2nd edition, Academic Press, Inc., 1983; And WadeLG, COMPENDIUM OF ORGANIC SYNTHETIC METHODS, John Wiley and Sons, 1980).

In some embodiments, R ⁴, R ⁴', R ⁵and R ⁵' independently selected from H, halogen, NH ₂, OMe, O (CH ₂) ₂n (Me) ₂and NO ₂.In some embodiments, R ⁴, R ⁴', R ⁵and R ⁵' at least one be O (CH ₂) ₂n (Me) ₂.In some embodiments, R ⁴, R ⁴', R ⁵or R ⁵' at least one be O (CH ₂) ₂n (Me) ₂, and other R ⁴, R ⁴', R ⁵and R ⁵' be H.In other embodiments, R ⁴o (CH ₂) ₂n (Me) ₂, and R ⁴', R ⁵and R ⁵' be H.

In some embodiments, R ⁷cH ₂-X ¹, wherein, X ¹f, Cl or Br, and R ⁶do not exist.In some embodiments, select medicine, make leaving group X ¹be selected from halogen, alkyl sulphonyl, aryl sulfonyl and azide.In some embodiments, X ¹cl or Br.

In some embodiments, Z is O.In some embodiments, X and Z are O.

In some embodiments, R ²h, methyl or cyano group, and R ¹, R ¹' and R ²' be H.In some embodiments, R ¹, R ¹', R ²and R ²' be H.In some embodiments, R ¹, R ¹' and R ²' be H.

In some embodiments, R ³it is following reactive group.

The preferred structural formula of formula (14) compound is as follows:

Another preferred embodiment of formula (14) compound is as follows:

Another preferred embodiment of formula (14) compound is as follows:

With

preferred duocarmycin SA and CBI conjugate

Peptide of the present invention, hydrazine or disulphide linking group can be for comprising duocarmycin SA or CBI analog as the conjugate of cytotoxic agent.Preferred conjugate of the present invention will further describe below.Unless stated otherwise, substituent group is as above definition in the part about cytotoxin, peptide linking group, hydrazine linking group and disulphide linking group.

a. peptide linking group conjugate

In preferred embodiments, the invention provides the peptide linking group conjugate with following structure:

Or

Wherein, X ¹it is halogen;

X is selected from O, S and NR ²³;

R ²³be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; With

R ⁴, R ⁴', R ⁵and R ⁵' independently selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, OR ¹⁵and O (CH ₂) _nn (CH ₃) ₂,

Wherein,

N is 1 to 20 integer; With

R ¹⁵and R ¹⁶independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted, wherein, R ¹⁵and R ¹⁶together with the nitrogen-atoms connecting with them, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms.

The limiting examples of such conjugate comprises following structure

With

Wherein, X ¹cl or Br;

Wherein, Ab is antibody or its fragment.

In a further preferred embodiment, the invention provides the conjugate with following structure:

Wherein, X ¹it is leaving group;

Z and X are independently selected from O, S and NR ²³,

R ³be selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, OR ¹⁵and O (CH ₂) _nn (CH ₃) ₂,

Wherein,

N is 1 to 20 integer;

R ¹⁵and R ¹⁶independently selected from H, replace or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted, wherein, R ¹⁵and R ¹⁶together with the nitrogen-atoms connecting with them, be optionally connected to form 4 to 6 yuan and replace or unsubstituted Heterocyclylalkyl ring system, this ring system optionally comprises two or more hetero atoms.

The limiting examples of such conjugate comprises following structure:

With

Wherein, each b is 0 to 20 integer independently, and Ab is antibody, or its fragment.

In another preferred embodiment, the invention provides and there is the peptide linking group conjugate that is selected from following structure:

Wherein, X ¹be Cl or Br, and Ab is antibody, or its fragment.

In other embodiment, the invention provides and there is the peptide linking group conjugate that is selected from following structure:

With

Wherein, X ¹be Cl or Br, and Ab is antibody, or its fragment.

In other embodiment, the invention provides the peptide linking group conjugate with following structure:

Wherein, X ¹be Cl or Br, and Ab is antibody, or its fragment.

b. hydrazine linking group conjugate

In preferred embodiments, the invention provides the hydrazine linking group conjugate with following structure:

In a further preferred embodiment, the invention provides the hydrazine linking group conjugate with following structure:

With

In another preferred embodiment, the invention provides and there is the hydrazine linking group conjugate that is selected from following structure:

With

Wherein, PEG is polyalkylene glycol moiety, and X ¹cl or Br.

In other preferred embodiment, the invention provides and there is the hydrazine linking group conjugate that is selected from following structure:

With

Wherein, X ¹be Cl or Br, and Ab is antibody, or its fragment.

In another embodiment, hydrazine linking group conjugate is selected from following structure:

With

c. disulphide linking group conjugate

In preferred embodiments, the invention provides the disulphide linking group conjugate with following structure:

With

The limiting examples of this structure comprises as follows:

With

Wherein, X ¹be Cl or Br, and Ab is antibody, or its fragment.

part

Part of the present invention is expressed as " X ⁴".In the present invention, X ⁴representative is selected from shielded reactive functional groups, not protected reactive functional groups, detectable labelling and targeting agent.Preferred part is targeting agent, for example antibody and fragment thereof.

In preferred embodiments, radicals X ⁴can be described as being selected from R ²⁹, COOR ²⁹, C (O) NR ²⁹and C (O) NNR ²⁹, wherein, R ²⁹be selected from and replace or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted heteroaryl.In another exemplary embodiment, R ²⁹be selected from H; OH; NHNH ₂;

with

Wherein, R ³⁰representative is by the replacement of reactive functional groups end-blocking or unsubstituted alkyl, by the replacement of functional group dead-end or unsubstituted heteroaryl.Above structure is as reactive protecting group, and this protecting group can as the amino acid side chain of antibody reacts, be connected to linking group-drug moiety by targeting agent thus with for example targeting agent.

targeting agent

Linking group arm of the present invention can be connected with targeting agent with cytotoxin, and the latter's selectivity is sent and is loaded to cell, organ or body area.Exemplary target, to the part of agent such as antibody (such as chimeric, humanized and people's antibody), receptor, agglutinin, sugar, antibody etc., is that this area is confessed, can be used for ad lib implementing the present invention.Other targeting agent comprises a compounds, and they do not comprise specific molecular identification motif, comprise macromole, such as PEG, polysaccharide, polyamino acid etc., and they increase cytotoxic molecular mass.Extra molecular mass affects cytotoxic pharmacokinetics, for example serum half-life.

In exemplary embodiment, the invention provides the conjugate of cytotoxin, linking group or cytotoxin-linking group and targeting agent, targeting agent is a kind of biomolecule, for example antibody, receptor, peptide, agglutinin, sugar, nucleic acid or its combination.Exemplary conjugate approach of the present invention is as above described in flow process.

Can be used for implementing biomolecule of the present invention can derive from any source.Biomolecule can be from natural origin separation, or can utilize synthetic method preparation.Protein can be natural protein or the protein of sudden change.Mutation effect can be undertaken by chemomorphosis, the sensing mutation in site or the means of other induced mutation well known by persons skilled in the art.Can be used for implementing protein of the present invention and for example comprise enzyme, antigen, antibody and receptor.Antibody can be polyclonal or monoclonal, but most preferably monoclonal.Peptide can be separated from natural origin with nucleic acid, or can originally synthesize wholly or in part.

In preferred embodiments, targeting agent is antibody or antibody fragment, and it is to select according to the specificity of the antigen that on interested target cell or target position place is expressed.Identified a lot of TS or antigen that other diseases is special, and the antibody of those antigens this tumor for the treatment of or Other diseases have also been used or have been proposed for.The known antibody of prior art can, for conjugate of the present invention, be particularly useful for the disease that treatment is relevant to target antigen.Antibody-linking group-drug conjugate of the present invention can targeting the limiting examples (and relevant disease) of target antigen comprise: Her2 (breast carcinoma), CD20 (lymphoma), EGFR (entity tumor), CD22 (lymphoma, comprise non_hodgkin lymphoma), CD52 (chronic lymphatic leukemia), CD33 (acute myelogenous leukemia), CD4 (lymphoma, autoimmune disease, comprise rheumatoid arthritis), CD30 (lymphoma, comprise non_hodgkin lymphoma), Muc18 (melanoma), integrin (entity tumor), PSMA (carcinoma of prostate, benign prostatic hyperplasia), CEA (colorectal carcinoma), CD11a (psoriasis), CD80 (psoriasis), CD23 (asthma), CD40L (immune thromobcytopenic purpura), CTLA4 (t cell lymphoma) and BLys (autoimmune disease, comprise systemic lupus erythematosus (sle)).

Identification division is in those embodiments of protein or antibody therein, protein can be by any available reactive peptide residue on protein surface by spacer groups arm and surface or autologous gathering monolayer (SAM) component is connected or connected.In preferred embodiments, reactive group is amine or carboxylate.In particularly preferred embodiments, reactive group is the ε-amido of lysine residue.In addition, these molecules can for example, be adsorbed on the surface of substrate or SAM by non-specific interaction (chemisorbed, physical absorption).

Antibody recognition part can be for discriminatory analysis thing, and they are protein, peptide, nucleic acid, sugar or micromolecule, for example medicine, herbicide, pesticide, industrial chemical and ammunition.Arousing antibody is well known to a person skilled in the art to the method for specific molecular activity.Referring to JIUYUE in 1992, within 15th, authorize the people's such as Feng No. 5/147th, 786, United States Patent (USP); Authorize the people's such as Stanker No. 5/334th, 528, United States Patent (USP) on August 2nd, 1994; On November 11st, 1997 is authorized A1-Bayati, the United States Patent (USP) of M.A.S. the 5/686th, No. 237 and authorize the people's such as Hoess No. 5/573rd, 922, United States Patent (USP) on November 12nd, 1996.For connecting antibody and surperficial method, be also known in the art.Referring to people such as Delamarche, Langmuir12:1944-1946 (1996).

Targeting agent can connect linking group of the present invention by any available reactive group.For example, can connection peptides by amine, carboxyl, sulfydryl or hydroxyl.A kind of like this group can be positioned at end or the interior location of peptide chain.For example, for example, by the reactive group on alkali (ring outer amine) or the available hydroxyl on sugar moieties (3 '-or 5 '-hydroxyl), can connect nucleic acid.Peptide and nucleic acid chains can be further derived in one or more positions, to connect suitable reactive group on chain.Referring to people such as Chrisey, Nucleic Acids Res.24:3031-3039 (1996).

When peptide or nucleic acid are minute period of the day from 11 p.m. to 1 a.m synthesizing wholly or in part, can be during building-up process binding reactive group or masked reactive group.The derivative monomer of process that is much suitable for binding reactive group in peptide and nucleic acid is all well known by persons skilled in the art.For example, referring to The PEPTIDES:ANALYSIS, SYNTHESIS, BIOLOGY, Vol.2: " SpecialMethods in Peptide Synthesis ", Gross, E.and Melenhofer, J., Eds., Academic Press, New York (1980).A lot of useful monomers are commercially available (Bachem, the companies such as Sigma).Then this masked group can be removed after synthetic and shelter, now it can with the compounds of this invention component reaction.

Exemplary nucleic acid target comprises the nucleic acid of fit, antisense compounds and formation triple helical to agent.Conventionally, the hydroxyl of saccharide residue, from the amino of alkali residue or the phosphoric acid oxygen of nucleotide as necessary chemical functional group, make the agent of ucleotides targeting and cytotoxin coupling.But, those skilled in the art will readily appreciate, and other " non-natural " reactive functional groups can utilize routine techniques to be additional on nucleic acid.For example, the hydroxyl of saccharide residue can utilize technical transform well known in the art for sulfydryl or amino.

Fit (or nucleic acid antibody) is strand or double-stranded DNA or the single stranded RNA molecule of binding specificity molecule target.The effect of generally speaking, fit by Inhibitory molecules target--for example protein--, be combined in the target set circulating in blood and play a role.Fit have a chemical functional group, thus can with cytotoxin covalent bonding, as described herein.

Although the association that various molecule targets can be special with the non-covalent property of fit generation, comprise small-molecule drug, metabolite, cofactor, toxin, saccharide medicine, nucleotide drug, glycoprotein etc., but generally speaking molecule target will comprise protein or peptide, comprise serum albumin, kassinin kinin, c20 compounds, cell surface molecule etc.Fit example comprises GileadShi antithrombase inhibitor GS 522 and derivant (Gilead Science, Foster City, Calif.) thereof.Separately see the people Proc.Natl.Acad.Sci.USA 90:3745-9 (1993) such as Macaya; The people such as Bock, the people such as Nature (London) 355:564-566 (1992) and Wang, Biochem.32:1899-904 (1993).

Specific biological molecules is to specific fitly can utilize technology known in the art to differentiate.Such as referring to people such as Toole, No. 92/14843rd, (1992) PCT communique; No. 91/19813, Tuerk and Gold (1991) PCT communique WO; No. 92/05285th, Weintraub and Hutchinson (1992) PCT communique and Ellington and Szostak, Nature346:818 (1990).In brief, these technology involve the mating reaction of molecule target and oligonucleotide random mixture conventionally.Fit-molecule target coordination compound is separated in the oligonucleotide never coordinating.By fit, from separated coordination compound, reclaim amplification.Repeat this circulation, to differentiate fit sequence molecule target to high affinity.

About the caused disease of inappropriate expression by gene, specificity prevents or the expression that reduces this genoid has represented desirable therapy.In principle, the generation of specific gene product can be suppressed, reduce or cut off by the hybridization of single chain deoxynucleotide or ribose Deoxydization nucleotide, the sequence that available sequences in these nucleotide and mRNA or front-mRNA process in necessary transcription is complementary, or with gene itself in sequence be complementary.This genetic control example is often called antisense or anti-gene inhibition.Extra effect, for example those alkylating agents of the present invention are given in the effect of puting together of alkylating agent and nucleic acid.

Antisense compounds is nucleic acid, is designed in conjunction with mRNA, makes mRNA lose ability, or prevents the generation of mRNA, and this mRNA is responsible for generating specific protein.Antisense compounds comprises antisense RNA or DNA, strand or two strands, oligonucleotide or their analog, they can with single mRNA specific hybrid, prevent transcribing of mRNA and/or translating of RNA processing and/or coded polypeptide, reduce thus separately the quantity of coded polypeptide (people such as Ching, Proc.Natl.Acad.Sci.U.S.A.86:10006-10010 (1989); The people such as Broder, Ann.Int.Med.113:604-618 (1990); The people such as Loreau, FEBS Letters 274:53-56 (1990); The people such as Holcenberg, WO 91/11535; WO 91/09865; WO 91/04753; WO 90/13641; WO 91/13080; WO 91/06629 and EP 386563).Due to their sharp target sensitivity and selectivity, antisense oligonucleotide can be used for delivering therapeutic agents to desired molecule target, for example cytotoxin of the present invention.

Other people are reported that nucleic acid can form and be combined with duplex DNA via triple helical, and inhibition is transcribed and/or DNA synthesizes.Triple helical compound (also referred to as three chain medicines) is such oligonucleotide, they are in conjunction with the sequence of double-stranded DNA, plan selectivity suppresses transcribing of Disease-causing gene, viral gene for example, for example HIV and herpes simplex virus, and oncogene, that is to say that the protein that they stop on nucleus produces.These medicines are directly in conjunction with the double-stranded DNA in cellular genome, form triple helical body, prevent that cell from preparing target protein.For example, referring to No. the 5th, 176,996, PCT communique WO 92/10590, WO 92/09705, No. 91/06626, WO and United States Patent (USP).Thereby cytotoxin of the present invention is also puted together with the nucleotide sequence that forms triple helical.

The locus specificity of nucleic acid (for example antisense compounds and triple helical medicine) is also subject to the appreciable impact of the modification of phosphodiester bond or the chemical modification of oligonucleotide end indistinctively.So these nucleic acid can be by chemical modification; Strengthen overall combination stability, increase the stability about chemical degradation, increase the speed that oligonucleotide transhipment enters cell, give molecule with chemical reactivity.Build the various universal methods that can be used for the nucleic acid of antisense therapy and be summarised in the people such as van derKrol, the people such as Biotechniques 6:958-976 (1988) and Stein, CancerRes.48:2659-2668 (1988).Therefore,, in exemplary embodiment, cytotoxin of the present invention is puted together by modification and the nucleic acid of phosphodiester bond.

And, carry cytotoxic fit, the antisense compounds of the present invention and triple helical medicine can also comprise nucleotide replacement, addition, leave out or be shifted, as long as retain and the specific hybrid of relevant target sequence or the association functional character as oligonucleotide.For example, some embodiment will adopt the similar thing of thiophosphate, they tolerate the Degradation of ribozyme more than their naturally occurring di-phosphate ester homologue, thereby expection has persistence in higher body and larger effect (such as referring to people such as Campbell, J.Biochem.Biophys.Methods 20:259-267 (1990)).The phosphoramidic acid ester derivant of oligonucleotide is the complementary polynucleotide of known combination also, have the ability of holding in addition covalently bound part, will be applicable to method of the present invention.Such as referring to people such as Froehler, Nucleic Acids Res.16 (11): 4831 (1988).

In some embodiment, fit, antisense compounds and triple helical medicine will comprise O-methyl ribonucleotides (No. 360609th, EP communique).Can also use chimeric oligonucleotide (people such as Dagle, Nucleic Acids Res.18:4751 (1990)).About some application, antisense oligonucleotide and triple helical can comprise the polyamide nucleic acid (people such as Nielsen, No. 90/15065, Science254:1497 (1991) and PCT communique WO) or other cationic derivative (people such as Letsinger, J.Am.Chem.Soc.110:4470-4471 (1988)).Other application can utilize such oligonucleotide, wherein one or more phosphodiester bonds by etc. row's group replace, key between the long nucleotide of 2-4 atom for example, as described in PCT communique WO92/05186 and No. 91/06556, or by the dimethoxym ethane group (people such as Matteucci, J.Am.Chem.Soc.113:7767-7768 (1991)) or amide group people such as (, Science 254:1497-1500 (1991)) Nielsen replace.

In addition, the present invention can adopt nucleotide analog, for example wherein sugar or alkali by chemical modification." analog " form of purine and pyrimidine be well known in the art those, they are much as chemotherapeutics.Exemplary and non-exhaustive list comprises 4-acetylcytosine, 5-(carboxyl hydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxyl methylamino methyl-2-thiouracil, 5-carboxyl methylamino methyluracil, dihydrouracil, inosine, N ⁶-isopentenyl gland purine, 1-methyladenine, 1-methyl pseudouracil, 1-methyl guanine, M1I, 2,2-dimethylguanine, 2-methyladenine, 2-methyl guanine, 3-methylcystein, 5-methylcytosine, N ⁶-methyladenine, 7-methyl guanine, 5-methylamino methyluracil, 5-methoxy amino methyl-2-thiouracil, β-D-MANNOSE base queosine, 5 '-methoxycarbonyl group methyluracil, 5-methoxyuracil, 2-methyl mercapto-N ⁶-isopentenyl gland purine, uracil-5-ethoxyacetic acid methyl ester, uracil-5-ethoxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-sulfo-cytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, methyl uracil, N-uracil-5-ethoxyacetic acid methyl ester, uracil-5-ethoxyacetic acid (v), pseudouracil, queosine, 2-sulfo-cytosine and 2,6-diaminopurine.In addition, the conventional alkali alkali of halo for example.In addition, the 2 '-furanose position on alkali can be replaced by uncharged bulky group.The example of uncharged bulky group comprises branched alkyl, sugar and side chain sugar.

The end modified technology that also provides use, can be used for puting together cytotoxin and nucleic acid, revises cell type specificity, pharmacokinetics, core permeability and the absolute cellular uptake speed of oligonucleotide drug.For example, it is known for comprising reactive group, at 5 ' and 3 ' end, replacing, and allows so covalently bound cytotoxin.For example, referring to OLIGODEOXYNUCLEOTIDES:ANTISENSE INHIBITORS OF GENE EXPRESSION, (1989) Cohen, Ed., CRC Press; PROSPECTIS FOR ANTISENSE NUCLEIC ACID THERAPEUTICSFOR CANCER AND AIDS, (1991), Wickstrom, Ed., Wiley-Liss; GENEREGULATION:BIOLOGY OF ANTISENSE RNA AND DNA, (1992), Ericksonand Izant, Eds., Raven Press and ANTISENSE RNA AND DNA, (1992), Murray, Ed., Wiley-Liss.About relating to the universal method of antisense compounds, referring to ANTISENSE RNA AND DNA, (1988), D.A.Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y..

detectable labelling

Specific markers or detectable group for the compounds of this invention and method are not generally key points of the present invention, as long as it can significantly not disturb activity or the practicality of the compounds of this invention.Detectable group can be any materials with detectable physics or chemical property.The detectable immunoassay field that is marked at of this class has been ripe, and generally speaking most of labellings that can be used for arbitrarily these class methods can be applicable to the present invention.Thereby labelling is the compositions that can be detected by spectrum, photochemistry, biochemistry, immunochemistry, electricity, optics or chemical means arbitrarily.Can be used for labelling of the present invention and comprise magnetic beads (DYNABEADS for example ^tM), fluorescein stain (such as fluorescein isothiocyanate, texas Red, rhodamine etc.), radioactive label (for example ³h, ¹²⁵i, ³⁵s, ¹⁴c or ³²p), enzyme (for example horseradish peroxidase, alkali phosphatase and the conventional enzyme of other ELISA) and colorimetric labelling, such as gold colloidal or coloured glass or plastic bead (such as polystyrene, polypropylene, latex etc.).

According to method well known in the art, labelling can be directly or indirectly and the compounds of this invention coupling.As implied above, can use various labellings, easy degree, stability requirement, the available instrumentation that the selection of labelling depends on required sensitivity, put together with compound and process regulation.

When the compounds of this invention is puted together mutually with detectable labelling, this labelling is preferably from radiosiotope, fluorescent agent, fluorescent agent precursor, chromophore, enzyme and combination thereof.The method that various groups is conjugated to antibody is well known in the art.For example, detectable labelling frequent and that antibody is puted together is enzyme, for example horseradish peroxidase, alkali phosphatase, beta galactosidase and glucoseoxidase.

Nonradioactive labeling often connects by indirect means.Generally speaking, make ligand molecular (biological example element) and conjugate component covalent bonding.Part is then for example, in conjunction with another kind of molecule (streptavidin), the latter be originally detectable or with signaling system covalent bonding, for example detectable enzyme, fluorescent chemicals or chemiluminescence compound.

The component of conjugate of the present invention can also directly be puted together with signal generation compound, for example, put together with enzyme or fluorogen.The Some Related Enzymes serving as a mark will be mainly hydrolytic enzyme, be definitely phosphatase, esterase or glycosidase, or oxidase, be definitely peroxidase.Fluorescent chemicals comprises fluorescein and derivant, rhodamine and derivant thereof, red sulphonyl, umbelliferone etc.Chemiluminescence compound comprises luciferin and 2,3-dihydro phthalazine diketone, for example luminol.About the comment of various operable Mk systems or signal generation system, referring to United States Patent (USP) the 4th, 391, No. 904.

The means of certification mark are well known to a person skilled in the art.Thereby for example, if labelling is radioactive label, detection means comprises scintillation counter or photograph film, as autoradiography.If labelling is fluorescent labeling, it can detect like this, with the optical excitation fluorescent dye of suitable wavelength, then detects gained fluorescence.Fluorescence can be with the naked eye when detecting, by photograph film, utilize electronic detectors, such as charge (CCDs) or photomultiplier tube etc.Similarly, enzyme labelling can detect like this, for enzyme provides suitable substrate, then detects gained product.Finally, simple colorimetric labelling can detect by observing the color relevant with labelling simply.Thereby in various dip rod algoscopys, the gold of puting together often presents pink, and various puted together beadlet presents the color of beadlet.

Fluorescent labeling is preferred at present, because their advantage is to need preventive measure seldom and be applicable to high flux shadowgraph technique (optical analysis comprises and analyzes the digitized of image in comprising the integrated system of computer) in operation.Preferred labelling has one or more following features conventionally: high specific when hypersensitivity, high stability, low background, low environment sensitivity and labelling.A lot of fluorescent labelinies are all commercially available: (the Saint Louis of SIGMA chemical company, MO), Molecular Probes (Eugene, OR), R & D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECHLaboratories, Inc. (Palo Alto, CA), Chem Genes Corp., AldrichChemical Company (Milwaukee, WI), Glen Research, Inc., GIBCOBRL Life Technologies, Inc. (Gaithersburg, MD), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland) and Applied Biosystems (Foster City, CA), and a lot of other commercial source known to the skilled.In addition, those skilled in the art will confirm how according to the specific suitable fluorogen of application choice, and if be not the facile words of commercial appearance, also can again synthesize necessary fluorogen or modify commercially available fluorescent chemicals by synthesizing mean, to obtain required fluorescent labeling.

Except micromolecule fluorogen, the artificial analog of naturally occurring fluorescin and this albuminoid also can be used for the present invention.This albuminoid is such as comprising cnidarian green fluorescent protein (people such as Ward, Photochem.Photobiol.35:803-808 (1982), the people such as Levine, Comp.Biochem.Physiol., 72B:77-85 (1982)), yellow fluorescin (the people such as Baldwin from vibrio fischeri kind, Biochemistry 29:5509-15 (1990)), perdinin-the chlorophyll belonging to from the Diniferida Symbiodinium (people such as Morris, Plant Molecular Biology 24:673-77 (1994)), phycobniliprotein (such as phycoerythrin and the phycocyanin) (people such as Wilbanks from ocean blue antibacterial (such as cyanobacteria Synechococcus (Synechococcus)), J.Biol.Chem.268:1226-35 (1993)) etc.

Generally speaking, before forming between cytotoxin and targeting agent (or other reagent) and optional spacer groups and connecting, will activate at least one chemical functional group.It will be recognized by those skilled in the art that various chemical functional groups comprise that hydroxyl, amino and carboxyl can activate by various standard methods and condition.For example, the hydroxyl of cytotoxin or targeting agent can be processed and be activated by phosgene, forms corresponding chloro-formate, or activates and form corresponding carbonic ester with p-nitrophenyl chloroformate ester

In exemplary embodiment, the targeting agent that the present invention uses comprises carboxyl functional group.Carboxyl can be activated, for example, change into corresponding acyl halide or active ester.This reaction can be carried out under different condition, and as at March, ibid, illustrated in 388-89 page.In exemplary embodiment, by carboxylic group, react and prepare acyl halide with oxalyl chloride.The reagent activating and the composite reaction of cytotoxin or cytotoxin-linking group arm, form conjugate of the present invention.It will be recognized by those skilled in the art, use carboxylic targeting agent only illustrative, the reagent that contains a lot of other functional groups also can be puted together with linking group of the present invention.

reactive functional groups

For the sake of clarity, follow-up discussion concentrates on the effect of puting together of cytotoxin of the present invention and targeting agent.A kind of embodiment of the present invention of take is example, and those skilled in the art easily derive other embodiment thus.Discussion concentrates on single embodiment and does not limit the present invention.

Exemplary compound of the present invention carries reactive functional groups, and it is generally positioned at and replaces or unsubstituted alkyl or assorted alkyl chain, allows them easily to connect another kind of group.The rotine positioning of reactive group is the terminal position of chain.

Can be used for implementing reactive group of the present invention and reactive species and be generally bioconjugates chemical field known those.Reactive functional groups can be protected or not protected, can change by the known method in organic synthesis field the protective nature of group.The desirable reactive species that can carry out with reactive cytotoxin analog is those that carry out under relative gentle condition at present.They include but not limited to nucleophilic displacement of fluorine (for example amine and alcohol and carboxylic acid halides, active ester reacts), electrophilic substitution (for example enamine reaction) and for example, to the addition of carbon-to-carbon and carbon-hetero atom multiple bond (Michael reacts, Diels-Alder addition).These and other useful reaction is for example discussed at March, ADVANCED ORGANIC CHEMISTRY, the 3rd edition, John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, AcademicPress, SanDiego, 1996 and the people such as Feeney, MODIFICATION OF PROTEINS, Advances in Chemistry Series, Vol.198, American ChemicalSociety, Washington, D.C., 1982.

Exemplary response type comprises the reaction of carboxyl and various derivants thereof, and carboxy derivatives includes but not limited to N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, carboxylic acid halides, acylimidazole, thioester, p-nitrophenyl ester, alkyl, thiazolinyl, alkynyl and aromatic ester.Hydroxyl can be converted into ester, ether, aldehyde etc.Haloalkyl is converted into new group, for example by with the reacting of amine, carboxylate anion, mercaptan anion, carbonium anion or alcoholates ion.Dienophile (for example maleimide) group participates in Diels-Alder reaction.Aldehydes or ketones base can be converted into imines, hydrazone, semicarbazones or oxime, or via mechanism such as Grignard addition or lithium alkylide additions.Sulfonic acid halide easily reacts with amine, for example, generate sulfonamide.Amine or sulfydryl are for example acidylate, alkanisation or oxidation.Utilize cycloaddition, acidylate, Michael addition etc., alkene can be converted into one group of new compound.Epoxide easily reacts with amine and hydroxy compounds.

Those skilled in the art will readily appreciate, and these keys much can be pressed variety of way and utilize various conditions to generate.About the preparation of ester, for example, referring to March, ibid, at 1157 pages; About the preparation of thioester, referring to March, ibid, at 362-363, and 491,720-722,829,941 and 1172 pages; About the preparation of carbonic ester, referring to March, ibid, at 346-347 page; About the preparation of carbamate, referring to March, ibid, at 1156-57 page; About the preparation of amide, referring to March, ibid, at 1152 pages; About the preparation of urea and thiourea, referring to March, ibid, at 1174 pages; About the preparation of acetal and ketal, referring to Greene etc., ibid, and at 178-210 page and March, ibid, at 1146 pages; About the preparation of acyloxy alkyl derivative, referring to prodrug: TOPICAL AND OCULAR DRUG DELIVERY, K.B.Sloan, ed., Marcel Dekker, Inc., New York, 1992; About the preparation of enol ester, referring to March, ibid, at 1160 pages; About the preparation of N-sulfimide hydrochlorate, referring to people such as Bundgaard, J.Med.Chem., 31:2066 (1988); About the preparation of anhydride, referring to March, ibid, at 355-56, and 636-37,990-91 and 1154 pages; About the preparation of N-acyl group amide, referring to March, ibid, at 379 pages; About the preparation of N-Mannich base, referring to March, ibid, 800-02 and 828 pages; About the preparation of methylol ketone ester, referring to people such as Petracek, Annals NY Acad.Sci., 507:353-54 (1987); About the preparation of disulphide, referring to March, ibid, at 1160 pages; Preparation about phosphonate ester and amido phosphonate.

Reactive functional groups can be not protected, and can be chosen such that so that they do not participate in or disturbance reponse not.Alternatively, by the existence of blocking group, can protective reaction functional group not participate in reaction.It will be appreciated by those skilled in the art that and how to protect specific functional group not disturb selected a set of reaction condition.About the example of useful blocking group, referring to people such as Greene, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

Conventionally, utilize the chemical technology of standard, make targeting agent and cytotoxin covalently bound by their chemical functional groups separately.Optionally, linking group or medicine and this reagent are by one or more spacer groups couplings.Spacer groups, when combining use, can be identical or different.

Generally speaking, before generating key between cytotoxin and reactive functional groups and optional spacer groups, at least one chemical functional group will be activated.Those skilled in the art will understand, various chemical functional groups--comprising hydroxyl, amino and carboxyl--can utilize various standard methods and condition to activate.In exemplary embodiment, the present invention comprises carboxyl functional group as reactive functional groups.Carboxyl can be activated as described above.

pharmaceutical preparation and administration

In another preferred embodiment, the invention provides pharmaceutical preparation, it comprises the compounds of this invention and pharmaceutically acceptable carrier.

Comprise that pharmaceutically acceptable carrier is delivered to patient as the compound as herein described of its addition salts or hydrate can utilize various route of administration or mode.Applicable route of administration includes but not limited to suction, transdermal, oral, rectum, through mucous membrane, enteral and parenteral, and parenteral comprises intramuscular, subcutaneous and intravenous injection.Preferably, comprising antibody or antibody fragment is parenteral as the conjugate of the present invention of targeting moiety, more preferably intravenous.

When for herein time, term " administration " intends contain directly all and indirectly send compound to the means at its predictive role position.

Compound as herein described or the acceptable salt of its pharmacy and/or hydrate can be individually dosed, with other the compounds of this invention administering drug combinations, and/or the cocktail form administration to combine with other therapeutic agent.Certainly, can treat disease will be depended in part degree with the selection of the therapeutic agent of the compounds of this invention co-administered.

For example, when when suffering from patient's administration of the morbid state being caused by the organism that depends on self-induction agent, the compounds of this invention can be with cocktail form administration, wherein contains and is used for the treatment of pain, infection and other symptom relevant with disease and the medicine of side effect.This class medicine is such as comprising analgesic, antibiotic etc.

When accepting patient's administration for the treatment of of cancer, compound can, with cocktail form administration, wherein contain anticarcinogen and/or supplementary hardening agent.Compound can also wherein contain the medicine of radiotherapy therapy side effect with cocktail form administration, such as antiemetic, radioprotector etc.

Can for example comprise with the supplementary hardening agent of the compounds of this invention co-administered tricyclic antidepressant (for example miboplatin is bright, desipramine, amitriptyline, clomipramine, trimeprimine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); Non-tricyclic antidepressant (for example Sertraline, trazodone and citalopram); Ca ^{+ 2}antagonist (for example verapamil, nifedipine, nitrendipine and caroverine); Amphotericin; Triparanol analog (for example tamoxifen); Anti-arrhythmic (for example quinidine); Antihypertensive (for example reserpine); Thiol depletion agent (for example buthionine sulfoximine); And calcium folinate.

Reactive compound of the present invention is with form administration own, or with pharmaceutical compositions administration, wherein reactive compound and one or more pharmaceutically acceptable carriers, excipient or mixing diluents.For pharmaceutical composition of the present invention, normally prepare in the usual way, use the upper acceptable carrier of one or more physiologys, comprise excipient and auxiliary agent, they are conducive to reactive compound to be processed into the prepared product that can pharmaceutically use.Suitable preparation depends on selected route of administration.

For mucosal, in preparation, use the penetration enhancer be applicable to the barrier that will permeate.This class penetration enhancer is well known in the art.

For oral administration, compound can be prepared at an easy rate like this, is about to reactive compound and is combined with pharmaceutically acceptable carrier well known in the art.This class carrier makes the compounds of this invention can be mixed with tablet, pill, lozenge, capsule, liquid, gel, syrup, serosity, suspensoid etc., oral for the patient that will treat.The pharmaceutical preparation orally using can obtain like this, mixes with solid excipient, optionally grinds gained mixture, and the mixture of processing granular adds applicable auxiliary agent if necessary, obtains tablet or lozenge core.Applicable excipient is filler definitely, and for example sugar, comprises lactose, sucrose, mannitol or Sorbitol; Cellulose prepared product, for example corn starch, wheaten starch, rice starch, potato starch, gelatin, Tragacanth, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).If necessary, can add disintegrating agent, for example crospolyvinylpyrrolidone, agar or alginic acid or its salt, for example sodium alginate.

For lozenge core provides applicable coating.For this reason, priming can be used, wherein arabic gum, Talcum, polyvinylpyrrolidone, Carbopol gel, Polyethylene Glycol and/or titanium dioxide can be optionally contained; Paint solution; With applicable organic solvent or solvent mixture.Can add dyestuff or pigment to tablet or lozenge core, for differentiating or distinguish different active compound doses combinations.

The pharmaceutical preparation that can orally use comprises the capsule of the applicable pushing of being made by gelatin, and the sealing soft capsule of being made as glycerol or Sorbitol by gelatin and plasticizer.The capsule that is applicable to pushing can contain the mixture of active component and following ingredients: filler, for example lactose; Binding agent, for example starch; And/or lubricant, for example Talcum or magnesium stearate; With optional stabilizing agent.In soft capsule, reactive compound can be to be dissolved or suspended in applicable liquid, for example fatty oil, liquid paraffin or liquid macrogol.In addition, can add stabilizing agent.All oral Preparations should be all the dosage that is suitable for this class administration.

About buccal administration, compositions can be taked the tablet prepared in the usual way or the form of dragee.

For inhalation, the suitable form with aerosol of compound discharges from pressue device or nebulizer used according to the present invention, wherein utilize applicable propellant, for example dichlorodifluoromethane, trichlorine fluomethane, dichlorotetra-fluoroethane, carbon dioxide or other applicable gas.The in the situation that of pressurised aerosol, by the valve that provides metering to discharge, can determine dosage unit.Be used in gelatine capsule in inhaler or insufflator and cartridge case and can be mixed with the mixture of powders that contains compound and applicable powder substrate, for example lactose or starch.

Compound can be mixed with by injecting for parenteral, for example, inject fast or continuous infusion.Injection is the preferred medication of the present composition.Injection preparation can be unit dosage forms, for example, in ampoule or multi-dose container, wherein add antiseptic.Compositions can be taked the form of suspensoid, solution or emulsion in oiliness or aqueous carrier, and can contain preparation reagent, for example suspending agent, stabilizing agent and/or dispersant, for example crospolyvinylpyrrolidone, agar or alginic acid or its salt, for example sodium alginate.

Parenteral comprises the aqueous solution of the reactive compound of water-soluble form with pharmaceutical preparation.In addition, the oily injection suspensoid that can suitably prepare reactive compound.Applicable lipophilic solvent or carrier comprise fatty oil, for example Oleum sesami, or synthetic fatty acid ester, for example ethyl oleate or triglyceride, or liposome.Aqueous injection suspension can contain the material that increases suspensoid viscosity, for example sodium carboxymethyl cellulose, Sorbitol or glucosan.Optionally, suspensoid can also contain applicable stabilizing agent or increase the reagent of compound dissolution degree, to prepare highly enriched solution.For injection, medicine of the present invention can be made into aqueous solution, preferably at the compatible buffer of physiology for example in Hanks ' s liquid, Ringer ' s liquid or normal saline buffer solution.

Alternatively, active component can be powder type, forms before use, for example aseptic pyrogen-free water with applicable excipient.

Compound can also be mixed with rectal compositions, and for example suppository or enema,retention, for example, contain conventional suppository base, for example cocoa butter or other glyceride.

Except previous formulations, compound can also be mixed with Drug Storage prepared product.This class durative action preparation can for example, by implanting or dermal delivery (subcutaneous or intramuscular), intramuscular injection or transdermal patch administration.For example, for example, thereby for example, compound can be prepared together with applicable polymerization or hydrophobic material (emulsion in acceptable oil) or ion exchange resin, or is mixed with microsolubility derivant, slightly soluble salt.

Pharmaceutical composition can also comprise applicable solid or gel phase carrier or excipient.The example of this class carrier or excipient includes but not limited to calcium carbonate, calcium phosphate, various sugar, starch, cellulose derivative, gelatin and polymer, for example Polyethylene Glycol.

Preferred pharmaceutical composition is the compositions of preparation for injecting, for example intravenous injection agent, and according to pharmaceutical composition 100% gross weight, it comprises the Drug Ligand conjugate of approximately 0.01% to approximately 100% weight.Drug Ligand conjugate can be antibody-cytotoxin conjugate, wherein, has selected antibody to take particular cancers as target

library

The library that also comprises within the scope of the present invention cytotoxin-linking group and medicine-linking group conjugate of cytotoxin of the present invention, cytotoxin and linking group.Exemplary library comprises at least 10 kinds of compounds, more preferably at least 100 kinds of compounds, and then more preferably at least 1,000 kind of compound, and then more preferably at least 100,000 kind of compound.The form in library is easily inquired about with regard to special properties, for example cytotoxicity, enzyme or the splitting action of other lytic reagent to linking group.Example form comprises chip format, microarray (microarray) etc.

Parallel or to combine synthetic main purpose be the library that generates different molecular, their share a common characteristic, are called in this manual scaffold.By replace different parts on each variable part of scaffold molecule, in library, developable amount of space has increased.Theoretical and modern medicine chemistry is advocated the concept in occupied space, and it is to measure the key factor of given compound to the effect of given biological target.By creating different molecular libraries, the space of institute's targeting of exploitation vast scale, the probability of developing efficient lead compound has increased dramatically.

Parallel synthesizing generally carries out on solid phase carrier, for example polymer resin.Scaffold or other applicable intermediate by cytotoxic compounds cleavable be connected on resin.React, to modify the scaffold being connected on resin.The variation of reagent and/or reaction condition produces structure diversity, and this is the quality place in each library.

Parallel the synthesizing of " little " molecule (the non-oligomer of molecular weight 200-1000) rarely had trial before nineteen ninety.Such as referring to people such as Camps, Annaks de Quimica, 70:848 (1990).Recently, it is synthetic like parallel (also the claiming " combination ") of the solid phase support of thing and some prostaglandins and β-rotary simulation thing that Ellmann discloses 11 kinds of benzodiazepines.These open source literatures are United States Patent (USP) the 5th for example, 288, No. 514.Parallel another part of synthetic relevant open source literature of micromolecule can be referring to United States Patent (USP) the 5th, 324, No. 483.This patent discloses has synthesized separately 4 to 40 kinds of compounds in 16 kinds of different scaffolds.The people such as Chen also apply organic synthesis strategy, utilize multistep process to synthesize non-peptide library (people such as Chen, J.Am.Chem.Soc., 116:2661-2662 (1994)) on polymer support.

Once prepare the library of unique compounds, just can use the library of self-induction agent as starting point, utilize method as herein described to prepare the library of immunoconjugates or antibody.

medicine box

On the other hand, the invention provides and contain one or more the compounds of this invention or compositions and about using the medicine box of the description of compound or compositions.In exemplary embodiment, the invention provides for puting together the medicine box of linking group arm of the present invention and another kind of molecule.Medicine box comprises linking group, with the description that linking group is connected with particular functional group.Medicine box also can comprise one or more cytotoxic drugs, targeting agent, detectable labelling, drug salts or buffer.Medicine box also can comprise container, and optionally one or more bottles, test tube, flask or syringe.Other kit form will be apparent for those skilled in the art institute, also belong to scope of the present invention.

purification

In another exemplary embodiment, the invention provides the method for separated part of the present invention-cytotoxic molecule target, it and X ligand ⁴in conjunction with.The method preferably includes and makes to comprise that the cellular preparations of target contacts with immobilization compound, generates coordination compound thus between receptor and immobilization compound.

Cytotoxin of the present invention can utilize any art-recognized means to be fixed on affinity carrier.Alternatively, cytotoxin can utilize one or more linking groups of the present invention to be fixed.

In another exemplary embodiment, the invention provides the protein affinity purification substrate that comprises linking group of the present invention.

The inventive method for separating of target will adopt one or more affinity chromatography technology conventionally.Affinity chromatography is utilized biomolecule or the recognition site with high selectivity of biopolymer to the chemical constitution of some support, can be effectively separated they.In document, there are a lot of articles, monograph and books about affinity chromatography, comprise the themes such as affinity chromatography carrier, crosslinked member, part and their preparation and purposes.The example of these lists of references comprises: Ostrove, Methods Enzymol.182:357-71 (1990); Ferment, Bioeng.70:199-209 (1990); The people such as Huang, J.Chromatogr.492:431-69 (1989); " Purification of enzymes by heparin-Sepharose affinitychromatography ", J.Chromatogr., 184:335-45 (1980); Farooqi, Enzyme Eng., 4:441-2 (1978); Nishikawa, Chem.Technol., 5 (9): 564-71 (1975); The people such as Guilford, PRACT.HIGH PERFORM.LIQ.CHROMATOGR, Simpson (ed.), 193-206 (1976); Nishikawa, Proc.Int.WorkshopTechnol.Protein Sep.Improv.Blood Plasma Fractionation, Sandberg (ed.), 422-35 (1977); " Affinity chroma tography ofenzymes ", Affinity Chromatogr., Proc.Int.Symp.25-38 (1977) is (Pub.1978); AFFINITY CHROMATOGRAPHY:A PRACTICALAPPROACH, the people such as Dean, (ed.), IRL Press Limited, Oxford, England (1985).Those skilled in the art have a large amount of instructions when exploitation adopts the specific affinity chromatography method of material of the present invention.

In the method, can use the affinity chromatography medium of various chemical constitutions as carrier.For example, agarose gel and cross-linked agarose gel can be used as carrier material, because their hydrophilic makes them relatively not have non-specific bonding.Other useful carrier for example comprises controlled cellular glass (CPG) beadlet, cellulose granules, polyacrylamide gel beadlet and the Sephadex being made by glucosan and epoxychloropropane ^tMgel beadlet.

the using method of medicine-ligand conjugates

Except above-mentioned composition and construct, the present invention also provides the several methods that can utilize the compounds of this invention and conjugate to put into practice.Use the method for medicine-ligand conjugates of the present invention to comprise: kill inhibition tumor cell or growth of cancer cells or copy, treat cancer, treatment cancer before symptom, kill or suppress to express the Growth of Cells of autoimmune antibody or copy, treat autoimmune disease, treatment infectious disease, stop tumor cell or cancer cell multiplication, cell proliferation, prevention autoimmune disease and prophylaxis against infection diseases that prophylaxis of cancer, prevention are expressed autoimmune antibody.These usings method comprise to the animal that has a demand uses the medicine-ligand conjugates of effective dose as mammal or people.The preferred part of a lot of usings method as herein described comprises antibody and antibody fragment, and it take specific tumors cell, cancerous cell or other target area is target.

Medicine-ligand complex of the present invention is used for the treatment of cancer, autoimmune disease and the infectious disease of animal.Provide by providing compositions in the acceptable mode of pharmacy to patient, and the present composition of pharmacy effective dose and treat compositions and the method for tumor.

The present invention is especially used for the treatment of cancer and for the propagation of inhibition tumor cell or cancerous cell in animal.Symptom before cancer or cancer, includes but not limited to that any disease or disease that tumor, transfer or the uncontrolled Growth of Cells of take are feature can be treated or prevent by being used medicine-ligand complex of the present invention.Complex delivering drugs is to tumor cell or cancerous cell.In one embodiment, ligand specificity ground in conjunction with or be joined to cancerous cell or the relevant antigen of tumor cell.Because it approaches part, medicine can be absorbed in tumor cell or cancerous cell by for example receptor-mediated endocytosis.Antigen can adhere to tumor cell or cancerous cell, or can be the extracellular matrix protein relevant to tumor cell or cancerous cell.Once enter cell, linking group just, by tumor cell or the relevant protease hydrolysis cracking of cancerous cell, discharges medicine thus.The medicine discharging is free diffusion then, and causes cytotoxicity.In alternative embodiment, medicine is cracking from medicine-ligand complex outside tumor cell or cancerous cell, and medicine infiltrates through cell subsequently.

Part can be attached to for example tumor cell or cancerous cell, the lip-deep tumor cell of tumor cell or cancerous cell or cancer cell antigen, or belong to tumor cell or the cancer cell antigen of the extracellular matrix protein relevant to tumor cell or cancerous cell.Part can be designed to that specific tumor cell or cancerous cell type are had to specificity.Therefore the tumor that, can effectively be treated or the type of cancer can be by selecting part to change.

Can by medicine-ligand conjugates as the cancer of target before the exemplary embodiment of symptom include but not limited to: change life, hypertrophy, abnormal development, colorectal polyp, actinic ketatosis, actinic cheilitis, human papillomavirus, white macula, lychen planus and BD.

Can as the cancer of target or the exemplary embodiment of tumor, be included but not limited to by medicine-ligand conjugates: pulmonary carcinoma, colon cancer, carcinoma of prostate, lymphoma, melanoma, breast carcinoma, ovarian cancer, carcinoma of testis, CNS cancer, renal cancer, kidney cancer, cancer of pancreas, gastric cancer, oral cancer, rhinocarcinoma, cervical cancer and leukemia.It is evident that to those skilled in the art, specific targeting part for conjugate can be selected, and makes it that drug targeting is delivered in the tumor tissues by this Drug therapy and (selects the targeting agent special to tumor specific antigen).The example of this targeting part is that prior art is known, and its limiting examples comprises the anti-Her2 that treats breast carcinoma, treats lymphadenomatous anti-CD 20, the anti-PSMA for the treatment of carcinoma of prostate, and treatment lymphoma comprises the anti-CD30 of non_hodgkin lymphoma.

In embodiments, the invention provides the method for cell killing.The method comprises to cell uses a certain amount of the compounds of this invention that is enough to kill described cell.In exemplary embodiment, by compound to there being patient's administration of this cell.In further exemplary embodiment, be administered for the growth that delays or stop the tumor that comprises cell (for example, cell can be tumor cell).For administration retarding of growing, the speed of growth of cell should be than the speed of growth before administration slowly at least 10%.Preferably, the speed of growth will delay at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or stop completely.

effective dose

Be applicable to pharmaceutical composition of the present invention and comprise such compositions, wherein contain the active component for the treatment of effective dose, namely effectively realize the amount of its expection object.The effective substantial amount of application-specific will especially be depended on to treated disease.Determining completely in those skilled in the art's limit of power, especially according to the detailed disclosed content of this paper of effective dose.

For any compound as herein described, treatment effective dose can be determined from cell culture algoscopy at first.Target plasma concentration will be such activity compound concentration, and it can suppress growth or the differentiation of cell.In preferred embodiments, cytoactive is suppressed at least 25%.Can induce at least about 50%, 75% or even 90% or the target plasma concentration of the higher inhibiting reactive compound of cytoactive be preferred at present.Can monitor the inhibition percentage of patient's cytoactive, the appropriateness of the plasma drug level being reached to assess, and can adjust up or down dosage, to reach required inhibition percentage.

As known in the field, for the mankind's treatment effective dose, also can determine from animal model.For example, can formulate people's dosage, to reach, have been found that the effective circulation composition of animal.As mentioned above, monitoring cyto-inhibition, and adjust up or down dosage, can adjust people's dosage.

Treatment effective dose can also be determined from the human data of the compound about the similar pharmacologically active of known performance.Relative bioavailability and effect that dosage used can the compound based on institute's administration be compared with known compound are adjusted.

Based on said method and other method well known in the art, adjust dosage to realize people's maximum effect completely in the limit of power of those of ordinary skill.

The in the situation that of topical, the systemic circulation concentration of the compound of institute's administration will not be particular importance.Under this class situation, by the object of compound administration, be to reach at regional area the concentration that effectively realizes Expected Results.

For the purposes in relating to the preventing and/or treating of disease of abnormal cell proliferation, the circulation composition of the compound of institute's administration is preferably approximately 0.001 μ M to 20 μ M, and approximately 0.01 μ M to 5 μ M is preferred.

Compound described herein to the dosage of patient's oral administration conventionally from about 1mg/ days to approximately 10,000mg/ days, more generally from about 10mg/ days to approximately 1,000mg/ days, the most conventionally from about 50mg/ days to about 500mg/ days.According to patient's body weight, typical dosage is from approximately 0.01 to about 150mg/kg/ days, more generally from approximately 0.1 to about 15mg/kg/ days, and the most conventionally from approximately 1 to about 10mg/kg/ days, for example 5mg/kg/ days or 3mg/kg/ days.

For other administering mode, the adjustment at dosage and interval can vary with each individual, and to provide, the compound of administration is to the effective blood plasma level of specific clinical indication of being treated.For example, in one embodiment, can be by relatively high concentration administration according to compound of the present invention, every day is repeatedly.Alternatively, may more preferably by minimum effective drug concentration, give the compounds of this invention, and use dosage regimen not too frequently.This will provide the therapeutic scheme matching with individual disease seriousness.

Utilize instruction provided herein, can plan effective therapeutic treatment scheme, neither can cause substantive toxicity, treat completely effectively again the clinical symptoms being showed by particular patient.This planning should involve consideration following factors and select modestly reactive compound: the toxicity curve of the existence of compound effect, relative bioavailability, weight in patients, adverse side effect and seriousness, preferred administering mode and selected medicine.

The following example is further set forth compound of the present invention, compositions and method.These embodiment only, for illustrating, do not limit invention required for protection.

Embodiment

materials and methods

In the following example, unless have contrary regulation, temperature with degree Celsius (℃) provide; Operation is carried out under room temperature or ambient temperature (being generally about 18-25 ℃); The evaporation of solvent utilizes rotary evaporator, under decompression (4.5-30mmHg conventionally), carries out, and bathes 60 ℃ at the most of temperature; The process of reaction is conventionally succeeded by TLC, and the response time is only for illustrating; Fusing point is not calibrated; Product performance is gratifying ¹h-NMR and/or microanalysis data; Yield is only for illustrating; Also use following conventional abbreviation: mp (fusing point), L (liter), mL (milliliter), mmol (mM), g (gram), mg (milligram), min (minute), LC-MS (liquid chromatograph-gas-phase spectrum) and h (hour).

¹h-NMR spectrum is measured on Varian Mercury 300MHz spectrometer, consistent with the structure of appointment.Chemical drifting is that the 1000000/umber (ppm) that departs from tetramethylsilane is reported.Electronic spraying mass spectrum records on Perkin Elmer Sciex API 365 mass spectrographs.Elementary analysis is by Robertson Microlit Laboratories, Madison, and NJ carries out.Flash chromatography silica gel is E.Me rck level (230-400 order).Inverse analysis type HPLC carries out on HP 1100 or Varian Pro Star 210 instruments, with Phenomenex Luna 5 μ m C-18 (2) 150mm * 4.6mm posts or VarianMicrosorb-MV 0.1 μ m C-18 150mm * 4.6mm post.Flow velocity is 1mL/min, and it is that gradient or 10% to 100% buffer B that 0% to 50% buffer B is gone through 15 minutes gone through the gradient of 10 minutes, and under 254nm, UV detects.Buffer A is 20mM ammonium formate+20% acetonitrile or the acetonitrile that contains 0.1% trifluoroacetic acid, and buffer B is 20mM ammonium formate+80% acetonitrile or 0.1% trifluoroacetic acid aqueous solution.Anti-phase preparation HPLC carries out on Varian ProStar 215 instruments, with Waters Delta Pak 15 μ m C-18300mm * 7.8mm posts.

embodiment 1: peptide linking group conjugate synthetic

1.1a synthetic method

Route map 1

Route map 2

Route map 3

Route map 4

Route map 5

Route map 6

1.1b compound 1:N-[2 '-(N '-tertbutyloxycarbonyl-amino)-ethyl] the tertiary fourth of-valine synthesizing of ester.

In DMF (10mL) solution of 2-(N-tertbutyloxycarbonyl-amino)-bromic ether compound (1g, 4.5mmole) and the valine tert-butyl ester (0.936g, 4.5mmole), add potassium carbonate (1.85g, 13.5mmole).The mixture so obtaining is stirred and spent the night at 100 ℃.Concentrated reaction mixture, and on silica gel with ethyl acetate/hexane (3/7) as eluant by residue purification by flash chromatography, obtain the title compound (0.16g, 12%) of oily. ¹h NMR (CDCl ₃) δ 0.94 (ft, 6H), 1.44 (s, 9H), 1.473 and 1.475 (2s, 9H), 1.88 (m, 1H), 2.51 (m, 1H), 2.78 (m, 2H), 3.11 (m, 1H), 3.22 (m, 1H), 3.39 and 4.13 (2bt, 1H), 5.00 (bs, 1H) ppm; LC-MS (ESI) 205 (M+H ⁺-112), 261 (M+H ⁺-Bu), 317 (M+H ⁺).

synthesizing of 1.1c compound 2:N-(2-aminoethyl)-valine.

Compound 1 (137mg, 0.43mmole) is at room temperature dissolved in to TFA/ dichloromethane solution (2mL, 1/1).The mixture so obtaining is at room temperature stirred to 30min.Concentrated reaction mixture, to dry, obtains the title compound (0.18g, 95%) of oily. ¹h NMR (CD ₃oD) δ 1.07 and 1.16 (2d, 6H), 2.35 (m, 1H), 3.2 (m, 1H), 3.38 (m, 4H) ppm; LC-MS (ESI) 217 (M+H ⁺).

synthesizing of 1.1d compound 3.

To maleic amide-dPEG ₄dichloromethane (1mL) solution that dropwise adds compound 2 (80.7mg, 0.16mmole) and diisopropylethylamine (55.5 μ L, 0.32mmole) in the dichloromethane of-NHS ester (61mg, 0.16mmole) (2mL) solution.The mixture so obtaining is stirred and spent the night.On Rotary Evaporators, remove desolventizing, and on silica gel with dichloromethane, then with the dichloromethane solution of 5% methanol, finally with 100% methanol as eluant by residue purification by flash chromatography, obtain the title compound (87mg, 97%) of colorless oil. ¹H NMR(CDCl ₃)δ1.08(dd，6H)， 2.25(m，1H)，2.49(t，2H)，2.52(t，2H)，3.10-3.79(m，25H)，6.82(s，2H)ppm；LC-MS(ESI)559(M+H ⁺)。

1.1e compound 4:Fmoc-Cit-PABOH's is synthetic.

To Fmoc-Cit-OH (1.0g, 2.52mmole) with 4-aminobenzyl alcohol (341mg, disposable 2-ethyoxyl-1-carbethoxyl group-1 that adds in dichloromethane 2.77mmole) (10mL) and methanol (5mL) solution, 2-dihydroquinoline [EEDQ] (1.24g, 5.04mmole).In the dark stir the mixture 16 hours.On Rotary Evaporators, remove desolventizing, and grind white solid with ether (100mL).Suspension supersound process 5min by gained, then places 30min.Filter and collect white solid, with ether washing, and vacuum drying (1.23g, 97%). ¹h-NMR (DMSO) δ 1.32 to 1.52 (m, 2H), 1.52 to 1.74 (dm, 2H), 2.86 to 3.06 (dm, 2H), 4.1 (M, 1H), 4.42 (d, 2H), 5.07 (t, 1H), 5.40 (bs, 2H), 5.97 (t, 1H), 7.19 to 7.95 (m, 12H), 8.10 (d, 1H), 9.97 (s, 1H) ppm; LC-MS (ESI) 503.1 (M+H ⁺).

1.1f compound 5:Fmoc-Cit-PABC-PNP's is synthetic.

To compound 4 (309mg, 0.62mmole) and p-nitrophenyl chloroformate ester (372mg, the disposable pyridine (100 μ L, 1.23mmole) that adds in oxolane 1.85mmole) (30mL) and 1-methyl-2-pyrrolidine (1mL) solution.The mixture so obtaining is at room temperature stirred 30 minutes.On Rotary Evaporators, remove desolventizing, and on silica gel with dichloromethane, follow the dichloromethane solution with 3% methanol, finally with the dichloromethane solution of 10% methanol, as eluant, residue is used on silica gel to purification by flash chromatography, obtain title compound white solid (97.9mg, 70%).LC-MS(ESI)668(M+H ⁺)。

1.1g compound 6:Fmoc-Lys (Boc)-PABOH's is synthetic.

According to the preparation method of above-claimed cpd 4, prepare compound 6, productive rate 98%. ¹h-NMR (DMSO) δ 1.40 (s, 9H), 1.38 (m, 2H), 1.50 to 1.74 (dm, 2H), 3.04 (t, 2H), 3.30 (q, 3H), 4.19 to 4.31 (m, 2H), 4.41 (d, 2H), 4.55 (s, 2H), 7.28 to 7.68 (m, 12H), 8.00 (d, 1H) ppm; LC-MS (ESI) 574 (M+H ⁺).

1.1h compound 7:Fmoc-Lys (Boc)-PABC-PNP's is synthetic.

According to the preparation method of above-claimed cpd 5, prepare compound 7, productive rate 70%. ¹H NMR(CDCl ₃)δ1.44(s，9H)，-1.49-1.60(m，6H)，1.73(m，1H)，2.00(m，1H)，3.11(m，1H)，3.20(bs，1H)，4.23(m，2H)，4.46(bs，2H)，4.67(bs，1H)，5.56(bs，1H)，7.28(m，2H)，7.36-7.41(m，6H)，7.59(m，4H)，7.76(d，2H)，8.26(dd，2H)，8.45(bs，1H)ppm；LC-MS(ESI)639(M+H ⁺-Boc)，684(M+H ⁺-Bu)，739(M+H ⁺)，778(M+K ⁺)。

1.1i compound 8:Boc-Val-Cit-OH's is synthetic.

In water (40mL) solution of citrulline (2.54g, 14.50mmole) and sodium bicarbonate (1.28g), add the Boc-Val-OSu (4.34g, 13.81mmole) being dissolved in dimethoxy-ethane (DME).In order to help mixture to dissolve, add oxolane (10mL).The mixture so obtaining is at room temperature stirred and spent the night.Add aqueous citric acid solution (15%, 75mL), and with 10%2-propanol/ethyl acetate (2 * 100mL) extraction mixture.With saline (2 * 150mL) washing organic layer, and on Rotary Evaporators, remove desolventizing.The white solid of vacuum drying gained 5 hours, then uses ether (100mL) to process.After simple supersound process and grinding, filter and collect white solid product (1.39g, 27%). ¹H NMR(CD ₃OD)δ0.91(dd，3H)，0.98(dd，3H)，1.44(s，9H)，1.70(m，2H)，1.87(m，2H)，2.02(m，2H)，3.11(t，2H)，3.89(t，1H)，4.39(q，1H)，8.22(d，1H)ppm；LC-MS(ESI)375(M+H ⁺)。

1.1j compound 9:Boc-Val-Cit-PABOH's is synthetic.

According to the preparation method of above-claimed cpd 4, prepare compound 9, productive rate 71%. ¹h NMR (CD ₃oD) δ 0.93 and 0.97 (2d, 6H), 1.44 (s, 9H), 1.58 (m, 2H), 1.75 (m, 1H), 1.90 (m, 1H), 2.05 (m, 1H), 3.10 (m, 1H), 3.19 (m, 1H), 3.91 (d, 1H), 4.52 (m, 1H), 5.25 (s, 2H), 7.40 (d, 2H), 7.45 (dd, 2H), 7.64 (d, 4H), 8.29 (dd, 2H) ppm; LC-MS (ESI) 480 (M+H ⁺).

1.1k compound 10:Boc-Val-Cit-PABC-PNP's is synthetic.

By the THF of Boc-Val-Cit-PABOH (178mg, 0.370mmole) (8mL) and CH ₂cl ₂(4mL) solution at room temperature stirs 3h with PNP chloro-formate (160mg, 0.80mmole) and pyridine (65 μ L, 0.80mmole).Ethyl acetate (100mL) and 10% aqueous citric acid solution (50mL) are added to reactant mixture, and with salt water washing organic layer, dry and concentrated, and with 5% methanol as eluant by residue on silica gel by purification by flash chromatography, obtain the white solid (165mg, 70%) of title compound. ¹H NMR(CD ₃OD)δ0.93(dd，3H)，0.97(dd，3H)，1.44(s，9H)，1.58(m，2H)，1.75(m，1H)，1.89(m，1H)，2.05(m，1H)，3.10(m，1H)，3.20(m，1H)，3.90(d，1H)，4.51(m，1H)，4.55(s，2H)，7.29(d，2H)，7.55(d，2H)ppm；LC-MS(ESI)545(M+H ⁺-Boc)，645(M+H ⁺)，667(M+Na ⁺)，683(M+K ⁺)。

1.1l compound 12a's is synthetic.

In ethyl acetate (5mL) suspension of compound 11 (20mg, 0.078mmole), be blown into HCl gas 20min (at this moment, suspension becomes clear and bright solution).Stirred reaction mixture 5min again, then enriched mixture, to dry, obtains the yellow solid (26mg, 100%) of title compound, and it is without being further purified just for next step.LC-MS(ESI)260(M+H ⁺-Cl)，295(M+H ⁺)。

1.1m compound 12b's is synthetic.

In ethyl acetate (5mL) suspension of compound 11 (20mg, 0.078mmole), be blown into HBr gas 20min (at this moment, suspension becomes clear and bright solution).Stirred reaction mixture 5min again, then enriched mixture, to dry, obtains the yellow solid (33mg, 100%) of title compound, and it is without being further purified just for next step.LC-MS(ESI)260(M+H ⁺-Br)，339(M+H ⁺)，341(M+H ⁺+2)。

1.1n compound 13b's is synthetic.

In DMF (2mL) solution of compound 12a (26mg, 0.078mmole), add 5-(2-dimethylamino-ethyoxyl)-benzofuran-2-carboxylic acid (44mg, 0.155mmole) and EDC (30mg, 0.155mmole).The mixture so obtaining is at room temperature stirred to 2h.Enriched mixture, and residue is dissolved in to H ₂o/CH ₃cN/TFA (4/1.5/0.5,6mL), and it is put into refrigerator 3h.Filter and collect yellow solid (35mg, 85%). ¹H NMR(CD ₃OD)δ2.67(s，3H)，3.01(s，6H)，3.34(m，2H)，3.63(ft，1H)，3.89(s，3H)，3.91(m，1H)，4.41(m，3H)，4.54(m，1H)，4.65(m，1H)，7.20(dd，1H)，7.36(d，1H)，7.54(s，1H)，7.59(d，1H)，7.73(bs，1H)，11.75(s，1H)ppm；LC-MS(ESI)490(M+H ⁺-Cl)，526(M+H ⁺)。

1.1o compound 13c's is synthetic.

To compound 12b (19mg, 0.0387) in DMF (2mL) solution, add 5-(2-dimethylamino-ethyoxyl)-benzofuran-2-carboxylic acid HBr salt (25mg, 0.0775mmole) with PS-carbodiimide (82mg, mmole/g:0.94,0.0775mmole).Stirring at room reactant mixture 24h.After filtration, concentrated filtrate, and residue is dissolved in to H ₂o/CH ₃cN/TFA (2/0.75/0.25,3mL), and it is put into refrigerator 3h.Filter and collect yellow solid dry, obtain title compound (18mg, 82%).LC-MS(ESI)490(M+H ⁺-Br)，570(M+H ⁺)，572(M+H ⁺+2)。

1.1p compound 14a's is synthetic.

At-78 ℃, in dichloromethane (4mL) suspension of compound 13a (48mg, 0.10mmole), add p-nitrophenyl chloroformate ester (80mg, 0.40mmole) and triethylamine (56 μ L, 0.40mmole).Mixture is slowly warming up to room temperature, and continues to stir again 30min.In reactant mixture, add compound N-Boc-N, N '-dimethyl-ethylenediamine (166mg, 0.80mmole), and stir and spend the night.Enriched mixture, and as eluant, residue is used on silica gel to purification by flash chromatography with the dichloromethane of 1.25% methanol, obtain the white solid (71mg, 100%) of title compound. ¹H NMRδ1.45-1.47(m，9H)，2.69(s，3H)，2.97(s，3H)，3.14-3.34(m，4H)，3.81-3.92(m，8H)，4.38-4.47(m，3H)，4.70(d，1H)，7.05(dd，1H)，7.11(d，1H)，7.45(s，1H)，7.48(d，1H)，7.99(s，1H)，10.43(s，1H)ppm。LC-MS(ESI)710(M-H ⁺)。

1.1q compound 14b's is synthetic.

At 0 ℃, in dichloromethane (2mL) suspension of compound 13b (48mg, 0.075mmole), add chloro-carbonic acid 4-nitro phenyl ester (80mg, 0.4mmole) and triethylamine (40mg, 0.4mmole, 56 μ L).Mixture is warming up to room temperature, and continues to stir again 6h.Evaporating solvent, and wash residue with ether, obtain intermediate.Intermediate is dissolved in to dichloromethane (2mL), and adds N-Boc-N in reaction solution, N '-dimethyl-ethylenediamine (44mg, 0.2mmole) and triethylamine (20mg, 0.2mmole, 28 μ L).The mixture so obtaining is at room temperature stirred and spent the night.Enriched mixture, and with ammonium formate (20mM, pH7.0) and acetonitrile, as eluant, on C-18 post, with HPLC, purify residue and obtain the white solid (31mg, 54%) of title compound.LC-MS(ESI)755(M+H ⁺)。

1.1r compound 14c's is synthetic.

At 0 ℃, to the CH of compound 13c (24mg, 0.04mmole) ₂cl ₂(2mL) in solution, add p-nitrophenyl chloroformate ester (64mg, 0.32mmole) and triethylamine (22 μ L, 0.16mmole).The reactant mixture so obtaining is at room temperature stirred to 18h.In reactant mixture, add N-Boc-N, N '-dimethyl-ethylenediamine (94mg, 0.50mmole), and continue to stir again 50min.Concentrated reaction mixture, and as eluant, on silica gel, with purification by flash chromatography residue, obtain the white solid (28mg, 83%) of title compound with the dichloromethane solution of 5% methanol.LC-MS(ESI)490，570，684(M+H ⁺-Boc)，784(M+H ⁺)，805(M+Na ⁺)，722(M+K ⁺)。

1.1s compound 15a's is synthetic.

Compound 14a (70mg, 0.10mmole) is dissolved in to trifluoroacetic acid (5mL), and mixture is at room temperature stirred to 30min, and be concentrated into dryly, product (72mg, 100%) is without being further purified just for next step.HPLC shows its > 95% purity. ¹H NMRδ2.64(s，3H)，2.93(s，3H)，3.19(s，3H)，3.30(t，1H)，3.79(s，3H)，3.85(s，3H)，3.81-3.85(m，1H)，4.27-4.49(m，3H)，4.59(d，1H)，4.68(d，1H)，6.97(dd，1H)，7.03(d，1H)，7.38(s，1H)，7.41(d，1H)，8.00(brs，1H)，10.61(br s，1H)ppm。LC-MS(ESI)612(M+H ⁺)，634(M+Na ⁺)。

1.1t compound 15b's is synthetic.

According to the preparation method of above-claimed cpd 15a, prepare compound 15b, productive rate 100%. ¹H NMR(CD ₃OD)δ2.69(s，3H)，2.76(s，3H)，2.83(bs，1H)，3.01(s，6H)，3.08(bs，1H)，3.24(bs，2H)，3.42(m，2H)，3.63(bs，3H)，3.74(bs，1H)，3.91(s，3H)，3.92(m，1H)，4.40(bs，2H)，4.57(bs，2H)，4.71(bs，1H)，7.22(bd，1H)，7.36(s，1H)，7.56(s，1H)，7.59(d，1H)，8.04(bs，1H)ppm；LC-MS(ESI)490，526，640(M+H ⁺)，678(M+K ⁺)。

1.1u compound 15c's is synthetic.

According to the preparation method of above-claimed cpd 15a, prepare compound 15c, productive rate 100%.LC-MS(ESI)490，570，684(M+H ⁺)，722(M+K ⁺)。

1.1v compound 16a's is synthetic.

In dimethyl formamide (the 200 μ L) solution of compound 5 (12.5mg, 0.019mmole) and compound 15a (10mg, 0.014), add triethylamine (6 μ L, 0.044mmole).The mixture so obtaining is at room temperature stirred and spent the night.In mixture, add ether (5mL), white solid is precipitated out from solution.Cross filter solid, and with dichloromethane, then with the dichloromethane solution of 1% methanol, the dichloromethane solution of the dichloromethane solution of 2% methanol, 3% methanol and the dichloromethane solution of last 4% methanol as eluant, on silica gel with this solid of purification by flash chromatography, obtain the white solid (8.7mg, 56%) of title compound.LC-MS(ESI)470，1112(M+H ⁺)，1134(M+Na ⁺)，1150(M+K ⁺)。

1.1w compound 16b's is synthetic.

In DMF (0.35mL) solution of compound 15b (5mg, 0.0056mmole), add compound 5 (3.8mg, 0.0056mmole) and DIEA (2 μ L, 0.011mmole).The mixture so obtaining is at room temperature stirred to 5h.Enriched mixture, and with the dichloromethane solution of 10% methanol as eluant, on silica gel, use purification by flash chromatography residue, obtain the solid (3mg, 45%) of title compound.LC-MS(ESI)490，526，1169(M+H ⁺)，1208(M+K ⁺)。

1.1x compound 16c's is synthetic.

According to the preparation method of above-claimed cpd 16b, prepare compound 16c, productive rate 50%.LC-MS(ESI)490，570，1212(M+H ⁺)，1250(M+K ⁺)。

1.1y compound 17a's is synthetic.

To the disposable piperidines (100 μ L) that adds in dimethyl formamide (the 500 μ L) solution of compound 16a (8.7mg, 0.008mmole).The mixture so obtaining is at room temperature stirred 20 minutes.On Rotary Evaporators, remove desolventizing, and be placed in fine vacuum 1.5h.Residue is dissolved in minimum dichloromethane (100 μ L), and adds hexane (3mL) in solution, white solid is separated out from solution, this solid of filtering, and dry (6.7mg, 96.7%).MS(ES)470，890.1(M+H ⁺)，912(M+Na ⁺)，928(M+K ⁺)。

1.1z compound 17b's is synthetic.

According to the preparation method of above-claimed cpd 17a, prepare compound 17b, productive rate 95%.LC-MS(ESI)947(M+H ⁺)。

1.1aa compound 17c's is synthetic.

According to the preparation method of above-claimed cpd 17a, prepare compound 17c, productive rate 95%.LC-MS(ESI)1015(M+H ⁺)。

1.1bb compound 18a's is synthetic.

To compound 17a (4.2mg, 0.005mmole) with compound 3 (2.64mg, the disposable PyBOP (3.7mg, 0.007mmole) that adds in dichloromethane 0.005mmole) (1mL) solution, then adds diisopropylethylamine (1 μ L).The mixture so obtaining is at room temperature stirred and spent the night.On Rotary Evaporators, remove desolventizing.With preparative HPLC, purify residue, obtain light brown solid (2.6mg, 38.7%).MS(ES)470，1431(M+H ⁺)，1453(M+Na ⁺)，1469(M+K ⁺)。

1.1cc compound 18b's is synthetic.

To compound 17b (2.2mg, 0.0025mmole) and compound 3, in dichloromethane (the 400 μ L) solution of 5% methanol, add HBTU (9mg, 0.0046mmole) and DIEA (1.4 μ L, 0.0046mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent, with 10mM ammonium formate and acetonitrile, as eluant, purification residue on half preparative HPLC, obtains the title compound (1.1mg, 30%) of oily.LC-MS(ESI)490，526，1488(M+H ⁺)，1527(M+K ⁺)。

1.1dd compound 18c's is synthetic.

To compound 17c (6.5mg, 0.0065mmole) with compound 3 (5.5mg, 0.0097mmole) in dichloromethane (0.5mL) solution of 5% methanol, add HBTU (3.7mg, 0.0097mmole) and DIEA (3.4 μ L, 0.0194mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane of 30% methanol on silica gel, obtains the title compound (4mg, 30%) of oily.LC-MS(ESI)1532(M+H ⁺)，1554(M+Na ⁺)，1570(M+K ⁺)。

1.2 containing synthetic containing the peptide linking group of duocarmycin SA of self sacrifice type spacer groups method

1.2a reacts A:

In suspension to alkanisation core 7mg in 2mL ethyl acetate, slowly by the dry gas stream of HBr, until form clear and bright solution, this needs about 15 minutes.Concentrated reaction mixture, and high vacuum dry is spent the night.

1.2b reacts B:

In the DMF suspension of the bromomethyl opened loop compound of preparing (bromo methyl secocompound), add EDC (10mg in steps A, 0.054mMoles) with 5-nitrobenzofuran carboxylic acid (12mg, 0.054mMoles), and stir 6 hours.Then in this reactant mixture, add ethyl acetate and saline.Be extracted with ethyl acetate after 3 times, the concentrated organic layer merging, and with MeOH/DCM (amount of MeOH is cumulative) through filtered through silica gel.With mass spectrum, confirm product, M+1=530.

1.2c reacts C:

With 2mL DCM, 200 μ L 1-propenol-3s and pyridine (the 21 μ L) solution of methyl piperazine phosgene (11mg, 0.054mMoles), protect 4 '-OH 2 hours.Use silica gel chromatography product, and with Mass Spectrometric Identification, MS+1=654.

1.2d reacts D:

Under 40PSI, by Pd/C hydrogenolysis 45 minutes in DCM/MeOH (2: 1), carry out the reduction of nitro.Filtration product, concentrated filtrate, and dry under fine vacuum.With mass spectral analysis, confirm product MS+1=, and just carry out next step without being further purified.

1.2e reacts E:

To the MeOH/DCM of above compound (18mg, 0.024mMoles) (2: 1,3mL) in solution, add Fmoc-Val-Citruline (29mg, 0.06mMoles), the mixture of gained is stirred 10 minutes, until all acid is all dissolved.The EEDQ that adds 15mg, 0.06moles, in the dark stirred reaction mixture spends the night.Then concentrated reaction mixture, rinses with diethyl ether, and purifies residue with anti-phase preparative HPLC, obtains product, and this product is identified with mass spectrum, M+1=1103.

1.2f reacts F:

Use the 1mL DMF solution of 5% piperidines carry out Fmoc protecting group go protect 10 minutes.Concentrated reaction mixture, then rinses solid residue with diethyl ether.With mass spectrum, confirm product, MS+1=880, and M+K=919.

1.2g reacts G:

In DMF (1.5mL) solution of the unhindered amina of preparing in step F, add Mal-(PEG) ₄-NHS-ester (20mg), and stirred reaction mixture 1hr.Concentrated, then with anti-phase preparative HPLC, purify, obtain 2.8mg (from alkanisation core, gross production rate is 11%), it is confirmed with mass spectrum, MS+1=2178, M+Na=1300, and M+K=1316.

the 1.3 peptide linking groups of puting together with Tubulysine A synthetic

Synthesizing and bonding shown in part can pass through with PEG and peptide linking group.

Intermediate and showing synthesizing hereinbefore of part-drug conjugate (its Chinese medicine is Tubulysine A) containing peptide linking group.This basic skills can be used for other medicines.

synthesizing of 1.4a peptide-linking group conjugate 111

synthesizing of 1.4b peptide-linking group conjugate 112

synthesizing of 1.4c peptide-linking group conjugate 113

synthesizing of embodiment 2:6-unit hydrazine linking group conjugate

the 2.1 two dimethylhydrazine linking groups of 6-unit of puting together with the derivative cytotoxin of duocarmycin SA synthetic

the synthetic route chart of 2.1a compound 109

synthesizing of 2.1b compound 110

At ice bath temperature, to Cbz-dimethyl propylene propylhomoserin (1g, 3.98mMoles) in the solution of 30mLDCM, add HOAT (catalytic amount, 0.25 equivalent), DIPEA (2.8mL, 16mmoles), then add 2-chloro-1,3-methylimidazole alkane hexafluorophosphate (CIP) (1.2g, 4.4mmoles).Then in this reactant mixture, add Boc-NN (Me) (643moles, 4.4mmoles).This reactant mixture of stirring at room spends the night.In this reactant mixture, add 10% citric acid solution (100mL), and extract with DCM.Water, then uses saturated sodium bicarbonate solution, then washes organic facies with water again.Then concentrated organic facies, and the hexane solution of the ethyl acetate constantly increasing by polarity, by silicagel column purification, obtains 860mg, 107 of 57% productive rate, and it uses Mass Spectrometric Identification, M+1=380, and M+NH ₄ ⁺=397.

Use the Pd/C in MeOH, by catalytic hydrogenation, remove Cbz protecting group, obtain compound 108, it is confirmed with MS.

To PNPC-1918 (10mg, 0.1mmoles), in the solution of 2mL DCM, dropwise add the solution of compound 108 (60mg, 0.25mmoles) in 8mL DCM, and stirred reaction mixture 2 days, until all raw materials all disappear.By short silica gel top, filter reactant mixture, then concentrated, and purify with anti-phase preparative HPLC, obtain 4.2mg compound 109.It is identified with mass spectrum, M+1=740.The Boc that carries out compound 109 with pure TFA goes to protect 20 minutes, obtains compound 110.Product is identified with mass spectrum, M+1=640.

synthesizing of 2.1c compound 111

Ma1-PEG4-1-Phenylethanone. and compound 110 (3mg .005mmoles) is mixed concentrated, and under fine vacuum dried overnight.In this mixture, add 1mL 5% acetic acid solution preparing the previous day, and pass through molecular sieve drying.Less than within 1 hour, just having formed hydrazone.After this, concentrated reaction mixture, and with anti-phase preparative HPLC (ammonium formate Ph=7) purification, obtain 2.8mg compound 111 (productive rate 60%).With this product of Mass Spectrometric Identification, MS+1=1129, M+NH ₄=1146, and M+K=1168

2.2 with tubulysin cytotoxin put together two-dimethyl 6-unit hydrazine linking group synthetic

With the similar method shown in embodiment 2.1 can be for the synthesis of the two dimethyl 6-s unit hydrazine linking group as compound in tubulysin A with medicine.

the 2.3 hydrazine linking groups of puting together with duocarmycin SA analog synthetic

In solution to bromomethyl opened loop compound (0.074mMoles) in 3mL DMF, add 5-acetyl group indole-2-carboxylic acid ester (30mg, 0.15mMoles) and EDC (28mg, 0.15mMoles), and the mixture of gained is stirred and spent the night.Concentrated reaction mixture, and purify by silica gel chromatography with the DCM solution of 5%MeOH, obtaining 29mg (productive rate 74%) product, it is confirmed with mass spectrum, M+1=523.

To synthetic compound in step C, in the solution of 5mL DCM and 300 μ L 1-propenol-3s, add methyl piperazine phosgene (22mg, 0.11mmoles) and pyridine 44 μ L.Stirring at room reactant mixture 5 hours.Concentrated, then with 5%MeOH/DCM, as eluant, by silica gel chromatography, purify, obtain the product (productive rate 73%) that 48mg wants.With mass spectrum, confirm product, M+1=650.

By above compound (8.2mg, 0.012mmoles) and the anhydrous DCM solution of 5% acetic acid of Ma1-PEG4-hydrazine at room temperature stir 20 minutes, follow evaporating solvent, and by anti-phase preparative HPLC, obtain the end product that 2.5mg wants by acetonitrile and ammonium formate buffered water, it is confirmed with mass spectrum, M+1=1063.

the ring formation speed of 2.4a dimethyl 6-unit hydrazine linking group

The duocarmycin SA analog of puting together with dimethyl 6-unit hydrazine linking group is cultivated 24 hours under pH7.4 in buffer, and through after a while, the cyclisation product that assessment generates from the cyclisation of hydrazine linking group, discharges free duocarmycin SA analog thus.

Under pH=7.4, through 24 hours, detect the minimum of cyclisation product, show that the 6-unit hydrazine linking group of this form has shown relatively slow ring formation speed.

the ring formation speed of 2.4b couple-dimethyl 6-unit hydrazine linking group

The duocarmycin SA analog of puting together with two dimethyl 6-unit hydrazine linking group is cultivated under pH7.4 in buffer, and through after a while, the cyclisation product that assessment generates from the cyclisation of hydrazine linking group, discharges free duocarmycin SA analog thus.

Together with-dimethyl linking group two with 6-unit, cyclization is quite rapid, in several minutes, is just over.Therefore it is faster that, the cyclisation velocity ratio of the first hydrazine linking group of two-dimethyl 6-does not comprise two-dimethyl 6 yuan of linking groups partly.

synthesizing of embodiment 3:5-unit hydrazine linking group conjugate

the synthetic method of 3.1 compounds 4

cbz-DMDA-2,2-dimethyl malonic acid (1)

In the 25mL flask of stirrer, thermometer and reflux condenser is housed, to 2,2-dimethyl-malonic acid (2.0gm, 0.0151moles), thionyl chloride (1.35ml, in THF 0.0182moles) (15ml) solution, add a DMF, and reactant mixture is heated to reflux 2 hours, be then cooled to room temperature.At 0 ℃, this reactant mixture is dropwise added in THF (5ml) solution of Cbz-DMDA (4gm, 0.0182moles) and triethylamine (4ml, 0.0287moles), and stir 30min at this temperature.Solvent removed in vacuo, is dissolved in 1N HCl (50ml) by residue, and extracts with DCM (2 * 25ml).The organic layer merging with 1N NaOH (2 * 25ml) extraction, the water layer merging with dense HCl acidify (pH < 1), with EtOAc (2 * 25ml) extraction, crosses MgSO ₄dry, filter also vacuum concentration and become canescence sticky solid, 3.44gm, productive rate 68%.With mass spectrum, confirm compound 1:m/z 337.0[M+1] ⁺, HPLC retention time: 3.77min (mass spectrum).

cbz-DMDA-2,2-dimethyl malonic acid-Boc-N '-methyl hydrazine (2)

In the 50mL 3N RBF flask of stirrer, thermometer and reflux condenser is housed, to compound 1 (3.0gm, 0.0089moles), thionyl chloride (0.78ml, in THF 0.0107moles) (25ml) solution, add a DMF, and make reaction mixture refluxed 2 hours, be then cooled to room temperature.Then this reactant mixture is dropwise added Boc-N-methyl hydrazine (1.33gm, 0.091moles) and triethylamine (3ml, 0.0215moles) at 0 ℃ in the solution of THF (25ml), and stir 30min.Solvent removed in vacuo, and residue is dissolved in to EtOAc (50ml), cross MgSO ₄it is dry,, filtration vacuum concentration become the oil of brown.This oil is dissolved in to EtOAc, and with column chromatography (100%EtOAc) purification, obtains the clear and bright oil of 3.45gm, 83% productive rate.With mass spectrum, confirm compound 2:m/z 465.2[M+1], HPLC retention time: 3.97min (mass spectrum)

dMDA-2,2-dimethyl malonic acid-BocN '-methyl hydrazine (3)

In MeOH (30ml) solution of compound 2 (0.5gm, 0.0011moles), add 10%Pd/C (15mg), and reaction is placed in to Parr hydrogenator 30 minutes.Filtration catalizer, vacuum concentrated filtrate, to clear and bright oil, generates compound 3 (0.38gm).With NMR ( ¹h, CDCl ₃) confirmation product: δ 1.45 (s, 15H) 2.45 (s, 3H) 2.85 (s, 6H), 3.16 (s, 3H) 4.64 (m, 1H) 10.6 (bs, 1H); NMR ( ¹³c, CDCl ₃) δ 24.1,28.57,35.15,35.58,36.66,47.01,48.51,81.11,155.17,173.56,176.24.

synthesizing of compound 4

Mixed compound 3 (50mg, 0.1513mmoles), PNPC-1918 (20mg, 0.0315mmoles) and DCM (5ml) in the 15ml RBF of stirrer is housed.Stir this solution 30 minutes, then add triethylamine (25 μ L, 0.1794mmoles), and stir fresh yellow solution 1hr.The oil of vacuum concentrated solution yellowly, and purify (100%DCM to 1: 1 EtOAc/DCM), generate the pale solid of compound 4,22mg, (84%) by column chromatography.With mass spectrum, confirm product: m/z 825.7[M+1] ⁺, HPLC retention time: 7.65min (mass spectrum).

synthesizing of 3.2 antibody-drug conjugates containing 5-unit hydrazine linking group

This route map has proved that antibody and linking group-medicinal composition put together.These methods are that pharmaceutical field is known.The example of other reactive site comprise maleimide, with the Haloacetamide of thiol reactant on part, the mercaptan reacting with disulphide on part, with the hydrazides of aldehyde on part and reactive ketone and N-Hydroxysuccinimide, the isocyanates, isothiocyanate and the acid anhydride that react with amino on part.

embodiment 4: synthetic containing the linking group conjugate of disulfide moieties

Route map 1

Route map 2

Route map 3

synthesizing of 4.1a compound 1.

To adding triton B (40% methanol solution, 1.08mL, 0.25mmole) containing in the flask of PEG4 (3.88g, 20mmole), after 15 minutes, then add tert-butyl acrylate (3.62mL, 24mmole).Stirring at room mixture overnight.Vacuum concentrated mixture as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the colourless oil (2.35g, 36%) of title compound. ¹H NMRδ1.45(s，9H)，2.5(t，2H)，3.65(m，18H)。

synthesizing of 4.1b compound 2.

In dichloromethane (10mL) solution of compound 1 (1.17g, 3.6mmole), add triethylamine (532 μ L, 4mmole) and methane sulfonyl chloride (309 μ L, 4mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the yellow oil (1.3g, 89%) of title compound. ¹H NMRδ1.43(s，9H)，2.48(t，2H)，3.07(s，3H)，3.62-3.70(m，14H)，3.76(m，2H)，4.37(m，2H)。

synthesizing of 4.1c compound 3.

In ethanol (10mL) solution of compound 2 (1.3g, 3.25mmole), add Hydrazoic acid,sodium salt (423mg, 6.5mmole).The mixture so obtaining is refluxed and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the yellow oil (1.01g, 90%) of title compound. ¹H NMRδ1.45(s，9H)，2.50(t，2H)，3.40(t，2H)，3.62-3.73(m，16H)。

synthesizing of 4.1d compound 4.

The H that contains to compound 3 (470mg, 1.35mmol) ₂in ether (5mL) solution of O (25 μ L), add triphenylphosphine (391mg, 1.48mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the yellow oil (325mg, 75%) of title compound. ¹H NMRδ1.45(s，9H)，2.24(bs，2H)，2.51(t，2H)，2.91(t，2H)，3.56(m，2H)，3.63-3.66(m，12H)，3.72(m，2H)。

synthesizing of 4.1e compound 5.

In methanol (10mL) solution of 3-mercaptopropionic acid (1.22g, 11.5mmole), add aldrithio1-2 (3.78g, 17.25mmole).The mixture so obtaining is at room temperature stirred 3 hours.Evaporating solvent as eluant, is used purification by flash chromatography residue with the hexane solution of 30% ethyl acetate on silica gel, obtains the title compound (2.44g, 98%) of oily. ¹H NMRδ2.8(t，2H)，3.05(t，2H)，7.14(m，1H)，7.67(m，2H)，8.48(m，1H)。

Compound 5b: ¹h NMR δ 1.43 (d, 3H), 2.61 (m, 1H), 2.76 (m, 1H), 3.40 (m, 1H), 7.17 (m, 1H), 7.66 (m, 2H), 8.45 (m, 1H).

synthesizing of 4.1f compound 6.

3-methylbenzothiazole iodide (1g, 3.6mmole) are dissolved in to 2N sodium hydrate aqueous solution (10mL), at 100 ℃, stir the mixture 6 hours, then with 6N aqueous hydrochloric acid solution, be acidified to pH4, and extract with diethyl ether.Cross Na ₂sO ₄dry organic layer, rotary evaporation in vacuo, and residue is dissolved in to methanol (10mL), and add compound 5a (776mg, 3.6mmole).Stirring at room mixture 1 hour.Enriched mixture, to dry, as eluant, is used purification by flash chromatography residue with the dichloromethane of 1% methanol on silica gel, obtains the yellow oil (482mg, 55%) of title compound. ¹H NMRδ2.85(m，2H)，2.95(m，5H)，6.64(m，2H)，7.3(m，1H)，7.4(dd，1H)；MS(ES)244(M+H ⁺)，487(2M+H ⁺)。

Compound 6b: ¹h NMR δ 1.35 (d, 3H), 2.48 (m, 1H), 2.92 (s, 3H), 3.02 (m, 1H), 3.34 (m, 1H), 6.62 (m, 2H), 7.28 (m, 1H), 7.44 (m, 1H); MS (ES) 258 (M+H ⁺).

Compound 6c: ¹h NMR δ 1.45 (s, 6H), 2.70 (s, 2H), 2.93 (s, 3H), 6.62 (m, 2H), 7.24 (m, 1H), 7.51 (m, 1H); MS (ES) 272 (M+H ⁺), 294 (M+Na ⁺), 310 (M+K ⁺).

synthesizing of 4.1g compound 7.

In absolute methanol (1mL) solution of compound 6a (28mg, 0.115mmole), add chloroacetic chloride (13 μ L, 0.173mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the hexane solution of 10% ethyl acetate on silica gel, obtains the title compound (24mg, 83%) of oily. ¹H NMRδ2.08(m，2H)，2.93(s，3H)，2.95(m，2H)，3.70(s，3H)，6.63(m，2H)，7.28(m，2H)，7.40(m，2H)；MS(ES)258(M+H ⁺)，280(M+Na ⁺)，296(M+K ⁺)。

Compound 7b: ¹h NMR δ 1.32 (d, 3H), 2.45 (m, 1H), 2.92 (s, 3H), 2.93 (m, 1H), 3.35 (m, 1H), 3.67 (s, 3H), 6.62 (m, 2H), 7.26 (m, 1H), 7.44 (m, 1H); MS (ES) 272 (M+H ⁺).

Compound 7c: ¹h NMR δ 1.42 (s, 6H), 2.66 (s, 2H), 2.93 (s, 3H), 3.62 (s, 3H), 6.62 (m, 2H), 7.24 (m, 1H), 7.51 (m, 1H); MS (ES) 286 (M+H ⁺), 308 (M+Na ⁺), 324 (M+K ⁺).

synthesizing of 4.1h compound 8.

At 0 ℃, in dichloromethane (1mL) solution of compound 7a (24mg, 0.093mmole), add triphosgene (28mg, 0.093mmole) and triethylamine (37 μ L, 0.28mmole).Stir the mixture 1 hour.Enriched mixture is to dry, and residue is without being further purified just for next step.

Coarse raw materials is dissolved in to dichloromethane (1mL), and adds compound 8a (35mg, 0.074mmole) and DMAP (23mg, 0.190mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the yellow oil (53mg, 76%) of title compound. ¹h NMR δ 2.70 (s, 3H), 2.74 (m, 2H), 3.06 (m, 2H), 3.34 (m, 1H), 3.35 and 3.36 (2s, 3H), 3.63 and 3.64 (2s, 3H), 3.86 (m, 1H), 3.88 (s, 3H), 3.93 and 3.94 (2s, 3H), 4.48 (m, 1H), 4.55 (m, 1H), 4.79 (m, 1H), 7.05 (m, 1H), 7.11 (m, 1H), 7.26-7.52 (m, 5H), 7.85 (d, 1H), 8.1 (bs, 1H), 8.98 and 9.08 (2s, 1H); MS (ES) 753 (M+H ⁺).

Compound 8b: ¹h NMR δ 1.38 (m, 3H), 2.52 (m, 1H), 2.69 (m, 3H), 2.79 (m, 1H), 3.33 (m, 1H), 3.37 (2s, 3H), 3.64 (m, 3H), 3.88 (s, 3H), 3.84-3.90 (m, 1H), 3.93 (2s, 3H), 4.48 (m, 1H), 4.57 (m, 1H), 4.78 (m, 1H), 7.06 (m, 1H), 7.12 (m, 1H), 7.26-7.43 (m, 3H), 7.50 (m, 2H), 7.86 (m, 1H), 8.1 (bs, 1H), 8.99,9.08,9.13 and 9.22 (4s, 1H); MS (ES) 767 (M+H ⁺).

Compound 8c: ¹h NMR δ 1.44 (m, 6H), 2.63 (d, 2H), 2.70 (s, 3H), 3.35 (m, 1H), 3.38 and 3.39 (2s, 3H), 3.63 and 3.64 (2s, 3H), 3.87 (m, 1H), 3.88 (s, 3H), 3.93 and 3.94 (2s, 3H), 4.48 (m, 1H), 4.55 (m, 1H), 4.79 (m, 1H), 7.05 (m, 1H), 7.12 (m, 1H), 7.31-7.39 (m, 3H), 7.49 (m, 2H), 7.89 (d, 1H), 8.1 (bs, 1H), 9.12 and 9.23 (2s, 1H); MS (ES) 781 (M+H ⁺).

4.1i compound 9 and 10 is synthetic.

In PBS buffer solution (pH7.2)/methanol (300 μ L, 2/1) solution of compound 8a (0.1mg), add 20mM DTT solution (100 μ L, 15 equivalents), with HPLC, monitor reaction process.Reaction is carried out too soon, to such an extent as to cannot detect, and after the several seconds, reaction completes, and has quantitatively generated compound 10.Reaction intermediate compound 9 do not detected.

4.1j compound 11 is synthetic.

In dichloromethane (1mL) solution of compound 6a (66mg, 0.2mmole), add DCC (47mg, 0.22mmole), HOBt (31mg, 0.22mmole) and compound 4 (50mg, 0.2mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the yellow oil (70mg, 62%) of title compound. ¹H NMRδ1.44(s，9H)，2.51(t，1H)，2.63(t，2H)，2.93(d，3H)，3.01(t，2H)，3.45(m，2H)，3.55(m，2H)，3.64(m，12H)，3.71(t，2H)，5.01(bs，1H)，6.38(bt，1H)，6.62(m，2H)，7.27(m，1H)，7.43(dd，1H)。MS(ES)491(M-56+H ⁺)，513(M-56+Na ⁺)，547(M+H ⁺)，569(M+Na ⁺)。

Compound 11b: ¹h NMR δ 1.34 (d, 3H), 1.45 (s, 9H), 2.30 (m, 1H), 2.5 (t, 2H), 2.69 (m, 1H), 2.93 (d, 3H), 3.37-3.55 (m, 5H), 3.63 (m, 12H), 3.71 (t, 2H), 4.99 (bs, 1H), 6.13 (bt, 1H), 6.62 (m, 2H), 7.25 (m, 1H), 7.48 (dd, 1H).MS(ES)505(M-56+H ⁺)，527(M-56+Na ⁺)，543(M-56+K ⁺)，561(M+H ⁺)，583(M+Na ⁺)。

Compound 11c:1.43 (s, 3H), 1.45 (s, 9H), 2.46 (s, 2H), 2.5 (t, 2H), 2.92 and 2.94 (2s, 3H), 3.33 (m, 2H), 3.47 (t, 2H), 3.63 (m, 12H), 3.70 (t, 2H), 6.06 (bt, 1H), 6.63 (m, 2H), 7.25 (m, 1H), 7.54 (d, 1H); MS (ES) 519 (M-56+H ⁺), 541 (M-56+Na ⁺), 575 (M+H ⁺), 597 (M+Na ⁺).

synthesizing of 4.1k compound 12:

At 0 ℃, to toluene (55 μ L, the 0.11mmole) solution that adds triethylamine (15 μ L, 0.11mmole) and 2N phosgene in dichloromethane (1mL) suspension of compound 11a (20mg, 0.037mmole).Stirring at room mixture 1 hour.Enriched mixture, and residue is dissolved in to dichloromethane (1mL), add compound 10 (14mg, 0.030mmole) and DMAP (9mg, 0.076mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent as eluant, is used purification by flash chromatography residue with the dichloromethane solution of 1% methanol on silica gel, obtains the yellow oil (23mg, 74%) of title compound. ¹h NMR δ 1.44 (s, 9H), 2.49 (t, 2H), 2.67 (m, 2H), 2.65 and 2.67 (2s, 3H), 3.07 (m, 2H), 3.33 (s, 3H), 3.40 (m, 3H), 3.51 (m, 2H), 3.60 (m, 12H), 3.69 (m, 2H), 3.87 (s, 3H), 3.92 (s, 3H), 3.93 (m, 1H), 4.52 (m, 2H), 4.78 (m, 1H), 6.65,6.74 and 6.97 (3bt, 1H), 7.06 (d, 1H), 7.12 (s, 1H), 7.29-7.42 (m, 3H), 7.50 (m, 2H), 7.87 (d, 1H), 8.10 and 8.15 (2bs, 1H), 9.79 and 9.58 (2s, 1H), MS (ES) 986 (M+H ⁺-56), 1042 (M+H ⁺).

Compound 12b: ¹h NMR δ 1.32 (m, 3H), 1.44 (s, 9H), 2.39 (m, 1H), 2.48 (m, 2H), 2.60 (m, 1H), 2.67 and 2.69 (2s, 3H), 3.32 and 3.35 (2s, 3H), 3.38-3.72 (m, 20H), 3.88 (s, 3H), 3.93 (s, 3H), 3.94 (m, 1H), 4.52 (m, 2H), 4.77 (m, 1H), 6.53,6.67 and 6.72 (3bt, 1H), 7.06 (d, 1H), 7.12 (s, 1H), 7.29-7.39 (m, 3H), 7.49 (m, 2H), 7.88 (d, 1H), 8.12 and 8.25 (2bs, 1H), 9.13,9.36,10.08 and 10.21 (4s, 1H), MS (ES) 1000 (M+H ⁺-56), 1056 (M+H ⁺), 1078 (M+Na ⁺), 1084 (M+K ⁺).

Compound 12c: ¹h NMR δ 1.30-1.42 (m, 3H), 1.44 (s, 9H), 2.45-2.52 (m, 4H), 2.69 and 2.72 (2s, 3H), 3.34 and 3.35 (2s, 3H), 3.39-3.72 (m, 19H), 3.88 (s, 3H), 3.925 and 3.93 (2s, 3H), 3.94 (m, 1H), 4.53 (m, 2H), 4.80 (m, 1H), 6.63 (m, 1H), 7.06 (dd, 1H), 7.13 (d, 1H), 7.25-7.39 (m, 3H), 7.50 (m, 2H), 7.89 (d, 1H), 8.10 and 8.27 (2bs, 1H), 9.99 and 10.191 (2s, 1H); MS (ES) 1014 (M+H ⁺-56), 1070 (M+H ⁺), 1108 (M+K ⁺).

4.1l compound 13 is synthetic.

By (1mL, 1/1) in the solution of compound 12a (23mg, 0.022mmole) solution trifluoroacetic acid and dichloromethane, stirring at room mixture 30min, the concentrated product (21mg, 100%) that obtains. ¹h NMR δ 2.60 (t, 2H), 2.67 and 2.68 (2s, 3H), 2.75 (m, 2H), 3.07 (m, 2H), 3.34 (s, 3H), 3.38-3.64 (m, 21H), 3.76 (t, 2H), 3.88 (s, 3H), 3.92 (s, 3H), 3.93 (m, 1H), 4.53 (m, 2H), 4.78 (m, 1H), 7.06 (d, 1H), 7.13 (s, 1H), 7.31-7.43 (m, 3H), 7.49 (m; 2H), 7.87 (d, 1H), 8.10 and 8.15 (2bs, 1H), 9.44 and 9.65 (2s, 1H); MS (ES) 986 (M+H ⁺), 1008 (M+Na ⁺), 1024 (M+K ⁺).

Compound 13b: ¹h NMR δ 1.34 (m, 3H), 2.56 (m, 1H), 2.62 (m, 2H), 2.68 (m, 3H), 2.8 (m, 1H), 3.35-3.36 (2s, 3H), 3.40-3.70 (m, 18H), 3.77 (t, 2H), 3.88 (s, 3H), 3.93 and 3.95 (2s, 3H), 3.94 (m, 1H), 4.54 (m, 2H), 4.79 (m, 1H), 7.07 (d, 2H), 7.13 (s, 1H), 7.30-7.42 (m, 3H), 7.49 (m, 2H), 7.88 (d, 1H), 8.11 and 8.25 (2bs, 1H), 9.22,9.37,9.80 and 9.92 (4s, 1H); MS (ES) 1000 (M+H ⁺), 1022 (M+Na ⁺), 1038 (M+K ⁺).

Compound 13c: ¹h NMR δ 1.30-1.45 (m, 6H), 2.54 (m, 2H), 2.61 (m, 2H), 2.68 and 2.69 (2s, 3H), 3.35-3.36 (2s, 3H), 3.40-3.70 (m, 17H), 3.77 (t, 2H), 3.88 (s, 3H), 3.92 and 3.93 (2s, 3H), 3.94 (m, 1H), 4.50 (m, 2H), 4.80 (m, 1H), 7.08 (m, 2H), 7.12 (d, 1H), 7.29-7.39 (m, 3H), 7.49 (m, 2H), 7.89 (m, 1H), 8.10 and 8.25 (2bs, 1H), 9.88 and 10.04 (2s, 1H); MS (ES) 1014 (M+H ⁺), 1036 (M+Na ⁺), 1054 (M+K ⁺).

4.1m compound 14a's is synthetic.

To compound 13a (5.4mg, in dichloromethane 0.0054mmole) (1mL) solution, add PS-carbodiimide (11.5mg, 0.94mmole/g, 0.0108mmole) and PS-DMAP (7.2mg, 1.49mmole/g, 0.0108mmole).The mixture so obtaining is at room temperature stirred and spent the night, filter and concentrate, obtain product.MS(ES)1082(M+H ⁺)。

the 4.2 disulphide linking groups of puting together with Tubulysin A synthetic

Use the mechanism showing above, medicine Tubulysin A can be conjugated to disulphide linking group of the present invention.Other medicines of the present invention and other linking group can be synthetic with similar reaction scheme figure.

the cyclisation speed of 4.3 disulphide linking groups

In PBS buffer (pH7.2)/methanol (300 μ L, 2/1) solution of compound 8a (0.1mg), add 20mM DTT solution (100 μ L, 15 equivalents), with HPLC, monitor reaction process.Quick cyclisation is carried out in reaction, within the several seconds, has reacted, and has quantitatively generated product 10.Reaction intermediate 9 do not detected.

embodiment 5

synthesizing of compound 32.

In ethyl acetate (10mL) solution of compound 30 (120mg, 0.28mmole), be blown into HCl gas 5min.Stirred reaction mixture 30min, then enriched mixture again under RT.Ether is added in reactant mixture, on filter funnel, collects white depositions.This solid of vacuum drying spends the night, and obtains the product that 100mg wants, and it is confirmed with LC-MS (ESI), 324 (M+H ⁺), and without being further purified just for next step.In DMF (5mL) solution of this compound (100mg, 0.24mmole), add compound 31 (65mg, 0.26mmole), HATU (100mg, 0.26mmole) and TEA (91 μ L, 0.52 mmole).The mixture so obtaining is at room temperature stirred to 3hrs.Evaporating solvent, with the solution of 0.1%TFA in water and acetonitrile, as eluant, purification residue on half preparative HPLC, obtains the compound 32 (110mg, 80%) of oily.With LC-MS (ESI), confirm the product of gained, 555 (M+H ⁺).

synthesizing of compound 33.

By the compound 32 (110mg, 0.2mmole) in DCM (10mL) and methanol (5mL) be adsorbed in palladium (20mg) stirring at room 12hrs under hydrogen-pressure of charcoal.Filter palladium, concentrated reaction mixture, uses the solution of 0.1%TFA in water and acetonitrile as eluant, purification residue on half preparative HPLC, the oily compound that obtains wanting (80mg, 78%).LC-MS(ESI)465(M+H ⁺)。At 0 ℃, to residue (80mg, in dichloromethane 0.17mmole) (10mL) and THF (5mL) solution, add PNPCl (chloro-carbonic acid 4-nitro phenyl ester) (137mg, 0.68mmole) and triethylamine (144 μ L, 1.02mmol).At 0 ℃, stir the mixture 30min so obtaining, then at room temperature stir 12hrs.Vacuum concentration reactant mixture, is settled out residue with ether (100mL), obtains the yellow oil (90mg, 82%) of compound 33, its vacuum drying, and with LC-MS (ESI), confirm 631 (M+H ⁺).

synthesizing of compound 46:

At room temperature in dichloromethane (10mL) solution of compound 33 (60mg, 0.1mmole), add Boc-N, N-dimethyl-ethylenediamine (84mg, 0.38mmole) and triethylamine (26 μ L, 0.1mmol).The mixture so obtaining is at room temperature stirred to 12hrs.Vacuum concentration reactant mixture, with ether (100mL) precipitation residue, obtains the compound 34 of Boc protection, and it is without being further purified just for next step.The compound 34 of Boc protection is dissolved in to 10mLTFA, reactant mixture is at room temperature stirred to 60min.Vacuum concentration reactant mixture, with ether (100mL) precipitation residue, obtains the yellow solid of compound 46, and it is by vacuum drying, and with LC-MS (ESI) confirmation, 631 (M+H ⁺).

synthesizing of compound 34.

In DMF (50mL) solution of 2-bromine ethamine bromide (5g, 24.4mmole), add diisopropylethylamine (8.5mL, 48.8mmole) and benzyl chloroformate (3.48mL, 24.4mmole).The mixture so obtaining is at room temperature stirred 2 hours.Concentrated reaction mixture, and on silica gel with ethyl acetate/hexane (3/7) as eluant by residue purification by flash chromatography, the compound 34 of the oily that obtains wanting (4g, 64%). ¹H NMR(CDCl ₃)δ3.54(bs，2H)，3.61(bs，2H)，5.12(s，2H)，7.36(m，5H)。

synthesizing of compound 35.

In DMF (50mL) solution of compound 34 (3.34g, 12.99mmole) and the valine tert-butyl ester (3.27g, 15.59mmole), add potassium carbonate (5.39g, 38.97mmole) and potassium iodide (2.59g, 15.59mmole).At 100 ℃, the mixture so obtaining is stirred and spent the night.Concentrated reaction mixture as eluant, is used purification by flash chromatography residue, the compound 35 of the oily that obtains wanting (3.12g, 69%) by ethyl acetate/hexane (2/8) on silica gel. ¹h NMR (CDCl ₃) δ 0.92 (m, 6H), 1.46 (s, 9H), 1.86 (m, 1H), 2.53 (m, 1H), 2.80 (m, 2H), 3.18 (m, 1H), 3.31 (m, 1H), 5.10 (s, 2H), 5.25 (bs, 1H), 7.36 (m, 5H); LC-MS (ESI) 296 (the M+H-tert-butyl groups ⁺), 352 (M+H ⁺).

synthesizing of compound 36.

Compound 35 (3.4g, 9.72mmole) in methanol (30mL) and palladium (200mg) room temperature that is adsorbed in charcoal are placed under hydrogen-pressure.The mixture so obtaining is at room temperature stirred 2 hours.Filter palladium, concentrated reaction mixture is extremely dry, the compound 36 of the oily that obtains wanting (2.1g, 98%).

synthesizing of compound 37.

At 0 ℃, in dichloromethane (30mL) solution of compound 36 (2.1g, 9.72mmole), add FmocOSu (9-fluorenylmethyloxycarbonyl-N-hydroxy-succinamide ester) (3.28g, 9.72mmole).At 0 ℃, the mixture so obtaining is stirred 2 hours.On Rotary Evaporators, remove desolventizing, and with dichloromethane, follow the dichloromethane solution with 0.5% methanol, finally use the dichloromethane solution of 1% methanol as eluant, on silica gel, use purification by flash chromatography residue, the colourless oil of the compound 37 that obtains wanting (2.55g, 60%). ¹h-NMR (CDCl ₃) δ 0.95 (ft, 6H), 1.48 (s, 9H), 1.90 (m, 1H), 2.55 (m, 1H), 2.82 (m, 2H), 3.18 (m, 1H), 3.32 (m, 1H), 4.24 (m, 1H), 4.37 (m, 2H), 5.40 (bs, 1H), 7.30 (m, 2H), 7.39 (m, 2H), 7.60 (d, 2H), 7.75 (d, 2H) ppm; LC-MS (ESI) 383 (the M+H-tert-butyl groups ⁺), 440 (M+H ⁺), 462 (M+Na ⁺), 478 (M+K ⁺).

synthesizing of compound 38.

To the oxolane-water of compound 37 (177mg, 0.4mmole) (3/1,8mL) in solution, be blown into HCl gas 5min.At 37 ℃, stirred reaction mixture spends the night, and then enriched mixture is extremely dry, the solid of the compound 38 that obtains wanting (168mg, 98%), and it confirms (ESI) 383 (M+H with LC-MS ⁺), 405 (M+Na ⁺), and without being further purified just for next step.LC-MS(ESI)383(M+H ⁺)，405(M+Na ⁺)。

synthesizing of compound 39.

In DMF (5mL) solution of compound 5 (525mg, 0.79mmole), add N-Boc-N, N '-dimethyl-ethylenediamine (177mg, 0.94mmole).The mixture so obtaining is at room temperature stirred to 30min.Except desolventizing, with dichloromethane, follow the dichloromethane solution with 2% methanol, finally use the dichloromethane solution of 5% methanol as eluant, on silica gel, use purification by flash chromatography residue, the colourless oil of the compound 39 that obtains wanting (364mg, 65%). ¹h NMR (CD ₃oD) δ 1.39 (s, 9H), 1.56 (m, 2H), 1.70 (m, 1H), 1.82 (m, 1H), 2.70 and 2.82 (2s, 3H), 2.90 (s, 3H), 3.09 (m, 1H), 3.17 (m, 1H), 3.30 to 3.37 (m, 4H), 4.16 (t, 1H), 4.27 (m, 1H), 4.33 (d, 2H), 5.02 (bs, 2H), 7.24 to 7.36 (m, 6H), 7.51 to 7.65 (m, 4H), 7.74 (d, 2H) ppm; LC-MS (ESI) 618 (M+H-Boc ⁺), 662 (the M+H-tert-butyl groups ⁺), 718 (M+H ⁺), 740 (M+Na ⁺), 1435 (2M+H ⁺).

synthesizing of compound 40.

According to the preparation method of above compound 17a, prepare compound 40, productive rate 98%.LC-MS(ESI)396(M+H-Boc ⁺)，496(M+H ⁺)，517(M+Na ⁺)，533(M+K ⁺)，992(2M+H ⁺)。

synthesizing of compound 41.

To compound 40 (138mg, in DMF 0.28mmole) (4mL) solution, add compound 38 (110mg, 0.28mmole), HOBt (36mg, 0.28mmole) and EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (50mg, 0.28mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent, with the water of 0.1%TFA and acetonitrile solution as eluant, purification residue on half preparative HPLC, the oil of the compound 41 that obtains wanting (178mg, 70%). ¹h NMR (CD ₃oD) δ 1.04 and 1.11 (2d, 6H), 1.40 (s, 9H), 1.58 (m, 2H), 1.77 (m, 1H), 1.88 (m, 1H), 2.24 (m, 1H), 2.72 and 2.84 (2s, 3H), 2.92 (s, 3H), 3.10 to 3.18 (m, 4H), 3.35 to 3.46 (m, 6H), 3.82 (d, 1H), 4.22 (t, 1H), 4.41 (m, 2H), 4.59 (m, 1H), 5.04 (bs, 2H), 7.28 to 7.40 (m, 6H), 7.55 (m, 2H), 7.63 (m, 2H), 7.78 (d, 2H) ppm; LC-MS (ESI) 760 (M+H-Boc ⁺), 804 (the M+H-tert-butyl groups ⁺), 860 (M+H ⁺), 882 (M+Na ⁺), 899 (M+K ⁺).

synthesizing of compound 42.

According to the preparation method of above compound 17a, prepare compound 42, productive rate 98%.LC-MS (ESI) 538 (M+H-Boc ⁺), 582 (the M+H-tert-butyl groups ⁺), 638 (M+H ⁺), 660 (M+Na ⁺).

synthesizing of compound 43.

At 0 ℃, to compound 42 (23mg, in dichloromethane 0.036mmole) (1mL) solution, add GMBS (N-(maleimide butyryl acyloxy) succinimide ester) (14mg, 0.05mmole) and diisopropylethylamine (8.4 μ L, 0.05mmole).Mixture is slowly warming up to room temperature, and continues to stir 30min.Evaporating solvent, uses 0.1%TFA water and acetonitrile solution as eluant, purification residue on half preparative HPLC, the compound 43 of the oily that obtains wanting (26mg, 79%). ¹h NMR (CD ₃oD) δ 1.06 and 1.12 (2d, 6H), 1.41 (s, 9H), 1.59 (m, 2H), 1.78 (m, 1H), 1.86 to 1.93 (m, 3H), 2.24 (m, 3H), 2.74 and 2.84 (2s, 3H), 2.93 (bs, 3H), 3.13 to 3.22 (m, 4H), 3.40 to 3.60 (m, 8H), 3.82 (d, 1H), 4.60 (m, 1H), 5.05 (bs, 2H), 6.80 (s, 2H), 7.32 (m, 2H), 7.57 (d, 2H), 8.78 (d, 1H) ppm; LC-MS (ESI) 703 (M+H-Boc ⁺), 747 (the M+H-tert-butyl groups ⁺), 803 (M+H ⁺), 825 (M+Na ⁺), 841 (M+K ⁺).

synthesizing of compound 44.

According to the preparation method of above compound 15a, prepare compound 44, productive rate 98%.LC-MS(ESI)703(M+H ⁺)，725(M+Na ⁺)。

synthesizing of compound 45.

At room temperature in DMF (0.8mL) solution of compound 44 (15mg, 0.016mmole) and compound 33 (10mg, 0.016mmole), add diisopropylethylamine (5.5 μ L, 0.032mmole).The mixture so obtaining is at room temperature stirred and spent the night.Evaporating solvent, with the water of 0.1%TFA and acetonitrile solution as eluant, purification residue on half preparative HPLC, the oily compound 45 that obtains wanting (10mg, 45%). ¹h NMR (CD ₃oD) δ 1.02 to 1.13 (m, 6H), 1.55 (m, 2H), 1.74 (m, 1H), 1.84 to 1.92 (m, 3H), 2.20 to 2.27 (m, 3H), 2.95 to 3.14 (m, 16H), 3.47 to 3.84 (m, 12H), 3.98 (m, 1H), 4.2 to 4.34 (m, 3H), 4.57 (m, 1H), 4.69 (m, 2H), 5.07 to 5.17 (m, 2H), 6.78 (s, 2H), 7.16 to 7.23 (m, 3H), 7.30 (m, 1H), 7.38 to 7.47 (m, 3H), 7.52 to 7.58 (m, 3H), 7.81 to 7.92 (m, 2H), 8.25 (bs, 1H) ppm; LC-MS (ESI) 1194 (M+H ⁺), 1215 (M+Na ⁺), 1233 (M+K ⁺).

embodiment 6

synthesizing of compound (2).

By MeOH/CH ₂cl ₂(1/2,1 (100mg, 0.24mmol) and 10%Pd-C (35mg) vacuum outgas 40s in 10ml).The mixture of gained is placed in nitrogen atmosphere, and stirs 7h at 25 ℃.By Celite (CH ₂cl ₂washed) filtration reactant mixture.Solvent removed in vacuo.With EtOAc/ hexane (2/8) elution on silica gel chromatography, obtain 2 (77mg, 98%). ¹NMR DMSO-d ₆δ10.36(s，1H)，8.04(d，1H，J＝8.2Hz)，7.72(d，1H，J＝8.2Hz)，7.61(brs，1H)，7.45(t，1H，J＝8.4Hz)，7.261(t，1H，J＝8.4Hz)，4.06(m，4H)，3.73(m，1H)，1.52(s，9H)。

synthesizing of compound (4).

At 25 ℃, in Ar, stir 4 M HC1-EtOAc (5ml) the solution 30min of 2 (35mg, 0.1mmol).Solvent removed in vacuo.In residue, add 5-acetyl indone-2-carboxylic acid (24.4mg, 0.12mmol).DMF (3ml) solution that adds EDC (22.9mg, 0.12mmol), stirred reaction mixture 5h at 25 ℃.Except desolventizing.CH with 10%MeOH ₂cl ₂solution elution is chromatography crude product on silica gel, obtains 4 (40.7mg, 93%). ¹HNMRDMSO-d ₆δ12.13(s，1H)，10.47(s，1H)，8.45(s，1H)，8.10(d，1H，J＝8.4 Hz)，7.96(brs，1H)，7.85(d，2H，J＝8.4 Hz)，7.54(d，1H，J＝8.4 Hz)，7.51(t，1H，J＝8.2Hz)，7.36(t，1H，J＝7.6)，7.35(s，1H)，4.81(t，1H，11.2 Hz)，4.54(dd，1H，8.8 Hz)，4.23(m，1H)，4.01(dd，1H，J＝10.2Hz)，3.86(dd，1H，J＝10.7Hz)，2.61(s，3H)。

synthesizing of compound (5).

By 4-methyl isophthalic acid-piperazine phosgene hydrochlorate (19.9mg, 0.1mmol) add 4 (20mg, 0.05mmol) and in anhydrous methylene chloride (4ml) solution of 3% 1-propenol-3 of anhydrous pyridine (25 μ ml, 0.3mmol), and the 16h that stirs the mixture.Purification of crude product on silica gel, obtains 5 (23.6mg, 91%). ¹NMR DMSO-d ₆)δ12.03(s，1H)，8.41(s，1H)，8.21(s，1H)，8.01(d，1H，J＝8.4 Hz)，7.88(d，1H，J＝8.4 Hz)，7.82(dd，1H，J＝8.4Hz)，7.58(t，1H，J＝8.1 Hz)，7.51(d，1H，J＝8.4Hz)，7.46(t，1H，J＝7.6Hz)，7.37(s，1H)，4.86(t，1H，J＝10.8Hz)，4.57(dd，1H，J＝10.8Hz)，4.38(m，1H)，4.06(dd，1H，J＝10.8Hz)，3.86(dd，1H，J＝11 Hz)，3.41(br，4H)，3.29(br，4H)，2.82(s，3H)，2.57(s，3H)。

synthesizing of compound (7).

At 25 ℃, by anhydrous methylene chloride (1ml) the solution stirring 30min of 5% acetic acid of 5 (13mg, 24 μ mol) and linking group 6 (16.9mg, 31 μ mol).Vacuum is thoroughly except desolventizing, with HPLC, purifies (SymmetryPrep C ₁₈, 7 μ m, 19 * 150mm post), obtain 7 (18.5mg, 81%).MS:C ₄₈h ₅₇clN ₈o ₁₁value of calculation (M+H) m/z 958.38, measured value 958.10.

embodiment 7: proliferation assay

The biologic activity of cytotoxic compound of the present invention can be used ripe ³h-thymidine proliferation assay is measured.This is a kind of method that is suitable for quantization cell propagation, because it is exogenous radiolabeled by measuring ³the combination of H-thymidine, evaluates DNA synthetic.This algoscopy is very reproducible, can adapt to a large amount of compounds.

When carrying out this mensuration, in the RPMI culture medium that contains 10% heat-inactivated hyclone (FCs), cultivate the leukaemia HL-60 of promyelocyte.Studying the same day, collecting cell, washing, by 0.5 * 10 ⁶the concentration of cell/mL is resuspended in the RPMI that contains 10%FCS.100 μ L cell suspensoids are joined in 96 hole flat boards.The serial dilution (3 times of increments) that carries out doxorubicin (as positive control) or test compound, every hole adds 100 μ L compounds.Finally, every hole adds the 100 μ Ci/mL of 10 μ L ³h-thymidine, cultivates flat board 24 hours.Utilize 96 hole harvesting devices (Packard Instruments) results dull and stereotyped, on Packard TopCount enumerator, count.According to four parameter logistic curves, measure ³the combination of H-thymidine, as the function of injection volume molar concentration, is used Prism software to measure IC ₅₀value.

The IC of the compounds of this invention in said determination method ₅₀value is generally from about 1pM to about 100nM, preferably from about 10pM to about 10nM.

embodiment 8: the puting together of medicine-linking group molecule and antibody

This embodiment described by medicine-linking group molecule of the present invention (optionally comprising other group, such as spacer groups and reactive functional groups etc.) with as the antibody X of targeting agent ⁴the reaction condition of puting together and method.These conditions and method are only intended to explanation, and unrestricted.Other method that medicine-linking group molecule and antibody are puted together is that prior art is known.

Conjugation methods described herein is that the lysine based on by antibody reacts with 2-iminothiolane, and then medicine-linking group molecule reacts with active maleimide base group, and free mercaptan group is introduced to antibody.At first, the antibody of puting together is cushioned fluid exchange to containing 50mM NaCl, 2mM DTPA, in the 0.1M phosphate buffer pH8.0 of pH8.0, and is concentrated into 5-10mg/ml.By adding 2-iminothiolane to realize mercaptan in antibody.In preliminary experiment, determine the amount of the 2-iminothiolane adding, and change to some extent according to the difference of antibody.In preliminary experiment, the 2-iminothiolane of cumulative amount is added dropwise to antibody, at room temperature with antibody, cultivate 1 hour subsequently, use Sephadex G-25 post that antibody is removed to freshen, put into the 50mM HEPES buffer of pH6.0, by reacting with dithio two pyridines (DTDP), the quantity of the thiol group that Fast Measurement is introduced.Thiol group and reacting of DTDP cause the release of the sulfo-pyridine that monitors at 324nm place.Use the sample that protein concentration is 0.5-1.0mg/ml.Protein concentration by the absorbance at 280nm place for Accurate Measurement sample, then at room temperature uses 0.1ml DTDP (alcoholic solution of 5mM storing solution) to cultivate every duplicate samples (0.9ml) 10 minutes.Independent buffer adds the also cultivation simultaneously of blank sample of DTDP.After 10 minutes, measure the absorbance at 324nm place, use 19800M ^-1the specific absorbance of sulfo-pyridine the quantity of the mercaptan existing is carried out quantitatively.

Conventionally, want the mercaptan level of three thiol groups of each antibody.For example, with a kind of specific antibody, by adding 15 times of molar excess, then at room temperature cultivate 1 hour, can realize this object.Therefore, with the mol ratio of wanting, cultivate the antibody that will put together with 2-iminothiolane, then desalination is assigned to and is puted together in buffer (50mM HEPES buffer, pH6.0, containing 5mM glycine, 3% glycerol and 2mM DTPA).Keep mercaptan raw material on ice, as mentioned above the mercaptan quantity of introducing is carried out quantitatively simultaneously.

After a certain amount of mercaptan has been introduced in evaluation, with the level of excessive 3 times moles of every part of mercaptan, add the medicine-linking group molecule containing active maleimide base group.Also comprise ultimate density be 5% glycol dimethyl ether (or suitable alternative solvent) put together buffer in carry out conjugation reaction.Conventionally, dissolved substance-linking group storing solution is in 90% glycol dimethyl ether, 10% dimethyl sulfoxine.For adding in antibody, storing solution can directly join in mercaptan antibody, this mercaptan antibody contains enough glycol dimethyl ethers, it is 5% that the amount of the glycol dimethyl ether adding makes its ultimate density, or beforehand dilution is in the puting together in buffer of the glycol dimethyl ether that is 10% containing ultimate density, then adds the mercaptan antibody of equal volume.

At room temperature stir, cultivate conjugation reaction 2 hours.After cultivating reaction, with 14000RPM, make mixture centrifugal 15 minutes, and by pH regulator to 7.2, if purification immediately not.Use several different methods by chromatography, to realize the purification of conjugate.Can, on the Sephacryl S200 post of crossing by the 50mM HEPES buffer balance of the pH7.2 containing 5mM glycine, 50mM NaCl and 3% glycerol in advance, use molecular-exclusion chromatography purification conjugate.Linear flow rate with 28cm/h carries out chromatography.Collect the part containing conjugate, mixed, and concentrated.Alternatively, can carry out purification by ion exchange chromatography.Condition changes according to the difference of antibody, and in each case, condition needs optimization.For example, antibody-drug conjugates reactant mixture is applied to the HEPES at 50mM, 5mM glycine, 3% glycerol, on the SP-Sepharose post that in pH6.0, pre-balance is crossed.With the 0-1M NaCl gradient elution antibody conjugates in level pad.The mixed part containing conjugate, by pH regulator to 7.2, and concentrating sample on demand.

Every part of patent application mentioning in this manual or relate to, patent, publication and other disclosed document are all incorporated herein by reference, and are equivalent to every part of independent patent application, patent, publication and other disclosed document and are all concrete and be introduced separately into herein as a reference.

Although described the present invention with reference to specific embodiments, but those skilled in the art should be understandable that, can carry out various changes, can replace equivalents, and not deviate from true spirit and the scope of the present invention and claims.In addition, can much revise, to adapt to specific situation, material, material composition, method, method step, adapt to object of the present invention, spirit and scope.All these classes are revised and are all intended to belong in the scope of claims.

Claims

1. following formula: compound

Wherein,

D has the drug moiety of structure below:

Wherein, ring system A is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl;

X is selected from O, S and NR ²³;

R ³be selected from (=O), SR ¹¹, NHR ¹¹and OR ¹¹,

Wherein,

R ⁴, R ^{4 '}, R ⁵and R ^{5 '}independently selected from the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO ₂, NR ¹⁵r ¹⁶, NC (O) R ¹⁵, OC (O) NR ¹⁵r ¹⁶, OC (O) OR ¹⁵, C (O) R ¹⁵, SR ¹⁵, OR ¹⁵, CR ¹⁵=NR ¹⁶and O (CH ₂) _nn (CH ₃) ₂

Wherein

N is 1 to 20 integer;

X ¹leaving group,

Wherein, R ¹¹, R ¹², R ¹³, R ¹⁵or R ¹⁶in at least one connect described drug moiety D to L ³, if present, or be connected to-NH-,

AA ¹represent single amino acids or a plurality of aminoacid that link together by amido link; Described aminoacid is natural amino acid and/or non-natural a-amino acid;

C is the integer between 1 to 20;

L ³it is the spacer groups that comprises primary amine or secondary amine or carboxyl functional group; Wherein, or L ³amine and the pendant carboxy group functional group of D form amido link, or L ³carboxyl and the suspension amine functional group of D form amido link;

O is 1;

L ⁴comprise polyalkylene glycol moiety;

Wherein, L ⁴do not comprise direct and (AA ¹) _cthe connected carboxylic acyl radical of N-terminal;

P is 1; With

X ⁴be selected from shielded reactive functional groups, not protected reactive functional groups, detectable labelling and targeting agent.

2. the compound of claim 1, wherein, polyalkylene glycol moiety comprises 3-12 recurring unit.

3. the compound of claim 2, wherein, polyalkylene glycol moiety comprises 2-6Ge recurring unit.

4. the compound of claim 3, wherein, polyalkylene glycol moiety comprises 4 recurring units.

5. according to the compound of claim 1 to 4 any one, wherein, (AA ¹) _cit is the peptide sequence of the protease cracking that can be expressed in tumor tissues.

6. the compound of claim 5, wherein, protease is lysosomal protein enzyme.

7. the compound of claim 1-4 any one, wherein, c is 2 or 3.

8. according to the compound of claim 1 to 4 any one, wherein, close to the (AA of drug moiety ¹) _cin aminoacid be selected from: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Va1.

9. according to the compound of claim 1 to 4 any one, wherein, (AA ¹) _cto be selected from following peptide sequence: Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N ⁹-tosyl-Arg, Phe-N ⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu and Gly-Phe-Leu-Gly.

10. according to the compound of claim 9, wherein, (AA ¹) _cval-Cit or Val-Lys.

The compound of 11. claim 1, it is selected from:

Wherein, X ¹be Cl or Br, and Ab is antibody, or its fragment.

12. 1 kinds of pharmaceutical preparatioies, compound and pharmaceutically acceptable carrier that it comprises claim 1-11 any one.